Mutant adeno-associated virus virions and methods of use thereof

ABSTRACT

The present invention provides mutant adeno-associated virus (AAV) that exhibit altered capsid properties, e.g., reduced binding to neutralizing antibodies in serum and/or altered heparin binding and/or altered infectivity of particular cell types. The present invention further provides libraries of mutant AAV comprising one or more mutations in a capsid gene. The present invention further provides methods of generating the mutant AAV and mutant AAV libraries, and compositions comprising the mutant AAV. The present invention further provides recombinant AAV (rAAV) virions that comprise a mutant capsid protein. The present invention further provides nucleic acids comprising nucleotide sequences that encode mutant capsid proteins, and host cells comprising the nucleic acids. The present invention further provides methods of delivering a gene product to an individual, the methods generally involving administering an effective amount of a subject rAAV virion to an individual in need thereof.

CROSS-REFERENCE

This application is a continuation-in-part of U.S. patent applicationSer. No. 10/880,297, filed Jun. 28, 2004, which claims the benefit ofU.S. Provisional Patent Application No. 60/484,111 filed Jun. 30, 2003,which applications are incorporated herein by reference in theirentirety.

FIELD OF THE INVENTION

The present invention is in the field of recombinant adeno-associatedvirus vectors.

BACKGROUND OF THE INVENTION

Adeno-associated virus (AAV) is a 4.7 kb, single stranded DNA virus thatcontains two open reading frames, rep and cap. The first gene encodesfour proteins necessary for genome replication (Rep78, Rep68, Rep52, andRep40), and the second expresses three structural proteins (VP1-3) thatassemble to form the viral capsid. As its name implies, AAV is dependentupon the presence of a helper virus, such as an adenovirus orherpesvirus, for active replication. In the absence of a helper itestablishes a latent state in which its genome is maintained episomallyor integrated into the host chromosome. To date, numerous AAV serotypesin humans have been identified.

In 1989 a recombinant AAV2 (rAAV) gene delivery vector system was firstgenerated, and vectors based on AAV have subsequently been shown tooffer numerous major advantages. First, vectors based on AAV areextremely safe, since wild-type AAV is nonpathogenic and has noetiologic association with any known diseases. In addition, AAV offersthe capability for highly efficient gene delivery and sustainedtransgene expression in numerous tissues, including muscle, lung, andbrain. Furthermore, AAV has enjoyed success in human clinical trials.

Despite this success, vector design problems remain. One major concernis the fact that much of the human population has already been exposedto various AAV serotypes, and as a result a significant fraction of anyfuture patient population harbors neutralizing antibodies (NABs) thatblock gene delivery. Additional problems with rAAV vectors includelimited tissue dispersion for serotypes that employ heparan sulfate as areceptor (AAV2 and 3), poor infection of non-permissive cell types suchas stem cells, challenges with high efficiency targeting of genedelivery to selected cell populations, and a finite transgene carryingcapacity.

There is a need in the art for improved AAV vectors that can infectcells that are non-permissive for AAV.

Literature

Halbert et al. (2000) J Virol 74, 1524-32; Blacklow et al. (1971) Am JEpidemiol 94, 359-66. (1971); Erles et al. (1999) J Med Virol 59,406-11; Moskalenko et al. (2000) J Virol 74, 1761-6; Wobus et al. (2000)J Virol 74, 9281-93; Sun et al. (2003) Gene Ther 10, 964-76; Nguyen(2001) Neuroreport 12, 1961-4; Davidson et al. (2000) Proc Natl Acad SciU S A 97, 3428-32; Rabinowitz et al. (1999) Virology 265, 274-85; Opieet al. (2003) J Virol 77, 6995-7006; U.S. Pat. No. 6,596,539; U.S. Pat.No. 6,733,757; U.S. Pat. No. 6,710,036; U.S. Pat. No. 6,703,237.

SUMMARY OF THE INVENTION

The present invention provides mutant adeno-associated virus (AAV) thatexhibit altered capsid properties, e.g., reduced binding to neutralizingantibodies in serum and/or altered heparin binding and/or alteredinfectivity of particular cell types. The present invention furtherprovides libraries of mutant AAV comprising one or more mutations in acapsid gene. The present invention further provides methods ofgenerating the mutant AAV and mutant AAV libraries, and compositionscomprising the mutant AAV. The present invention further providesrecombinant AAV (rAAV) virions that comprise a mutant capsid protein.The present invention further provides nucleic acids comprisingnucleotide sequences that encode mutant capsid proteins, and host cellscomprising the nucleic acids. The present invention further providesmethods of delivering a gene product to an individual, the methodsgenerally involving administering an effective amount of a subject rAAVvirion to an individual in need thereof.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1a and 1b depict heparin binding characteristics of wild type AAVversus the viral library.

FIGS. 2a and 2b depict generation of antibody neutralization escapemutants. FIG. 2a depicts the fraction rescued (normalized with respectto zero stringency) at various stringencies (given as the ratio ofneutralizing antibody titer (reciprocal dilution) divided by the actualdilution). FIG. 2b depicts the percent knockdown (reduction) ininfection by rabbit antisera for wild type AAV, AAV library, andindividual AAV escape mutants.

FIGS. 3A-C depict the nucleotide sequence of wild-type AAV cap (SEQ IDNO:1) aligned with nucleotide sequences of cap of the neutralizingantibody escape mutants AbE2 (SEQ ID NO:2) and AbE L (SEQ ID NO:3).Boxes indicate changes in nucleotide sequence compared to wild-type.

FIGS. 4A-G depict an alignment of VP1-encoding nucleotide sequences ofwild-type AAV-2 VP1 (SEQ ID NO:4), and exemplary neutralizing antibodyevasion mutants.

FIGS. 5A-C depict an alignment of VP1-encoding amino acid sequences ofwild-type AAV-2 VP1 (SEQ ID NO:5), and exemplary neutralizing antibodyevasion mutants.

FIGS. 6A-J depict an alignment of VP1-encoding nucleotide sequences ofwild-type AAV-2 VP1, and exemplary neutralizing antibody evasionmutants.

FIGS. 7A-D depict an alignment of VP1-encoding amino acid sequences ofwild-type AAV-2 VP1, and exemplary neutralizing antibody evasionmutants.

FIGS. 8A-G depict an alignment of VP1-encoding nucleotide sequences ofwild-type AAV-2 VP1, and exemplary heparin binding mutants.

FIGS. 9A-C depict an alignment of VP1-encoding amino acid sequences ofwild-type AAV-2 VP1, and exemplary heparin binding mutants.

FIGS. 10A and 10B provide an alignment of VP1-encoding amino acidsequences of wild-type AAV-5 and AAV2.5T, a capsid variant that confersincreased infectivity of lung epithelial cells.

FIGS. 11A-E depict the infectivity and the transduction of AVV vectorsfor pulmonary gene delivery in well-differentiated human airwayepithelia.

FIGS. 12A-D depict binding and cell specificity of an exemplary mutantinfectious for lung tissue.

FIG. 13 depicts infectivity of human embryonic stem cells of wild-typeAAV and AAV comprising variant capsid protein.

FIGS. 14A-D provide an alignment of amino acid sequences of exemplaryAAV capsid variants.

FIG. 15 depicts infectivity of human embryonic stem cells of wild-typeAAV and AAV comprising variant capsid protein.

FIGS. 16A-C provide an alignment of amino acid sequences of exemplaryAAV capsid variants.

FIGS. 17A-B provide an alignment of amino acid sequences of exemplaryAAV capsid variants hEr2.4, hEr1.23, and hEr3.1, compared to AAV2.

FIG. 18 depicts infectivity of human embryonic stem cells of wild-typeAAV and AAV comprising variant capsid protein.

DEFINITIONS

A “vector” as used herein refers to a macromolecule or association ofmacromolecules that comprises or associates with a polynucleotide andwhich can be used to mediate delivery of the polynucleotide to a cell.Illustrative vectors include, for example, plasmids, viral vectors,liposomes, and other gene delivery vehicles.

“AAV” is an abbreviation for adeno-associated virus, and may be used torefer to the virus itself or derivatives thereof. The term covers allsubtypes and both naturally occurring and recombinant forms, exceptwhere required otherwise. The abbreviation “rAAV” refers to recombinantadeno-associated virus, also referred to as a recombinant AAV vector (or“rAAV vector”). The term “AAV” includes AAV type 1 (AAV-1), AAV type 2(AAV-2), AAV type 3 (AAV-3), AAV type 4 (AAV-4), AAV type 5 (AAV-5), AAVtype 6 (AAV-6), AAV type 7 (AAV-7), AAV type 8 (AAV-8), avian AAV,bovine AAV, canine AAV, equine AAV, primate AAV, non-primate AAV, andovine AAV. “Primate AAV” refers to AAV that infect primates,“non-primate AAV” refers to AAV that infect non-primate mammals, “bovineAAV” refers to AAV that infect bovine mammals, etc.

An “rAAV vector” as used herein refers to an AAV vector comprising apolynucleotide sequence not of AAV origin (i.e., a polynucleotideheterologous to AAV), typically a sequence of interest for the genetictransformation of a cell. In general, the heterologous polynucleotide isflanked by at least one, and generally by two AAV inverted terminalrepeat sequences (ITRs). The term rAAV vector encompasses both rAAVvector particles and rAAV vector plasmids.

An “AAV virus” or “AAV viral particle” or “rAAV vector particle” refersto a viral particle composed of at least one AAV capsid protein(typically by all of the capsid proteins of a wild-type AAV) and anencapsidated polynucleotide rAAV vector. If the particle comprises aheterologous polynucleotide (i.e. a polynucleotide other than awild-type AAV genome such as a transgene to be delivered to a mammaliancell), it is typically referred to as an “rAAV vector particle” orsimply an “rAAV vector”. Thus, production of rAAV particle necessarilyincludes production of rAAV vector, as such a vector is contained withinan rAAV particle.

“Packaging” refers to a series of intracellular events that result inthe assembly and encapsidation of an AAV particle.

AAV “rep” and “cap” genes refer to polynucleotide sequences encodingreplication and encapsidation proteins of adeno-associated virus. AAVrep and cap are referred to herein as AAV “packaging genes.”

A “helper virus” for AAV refers to a virus that allows AAV (e.g.wild-type AAV) to be replicated and packaged by a mammalian cell. Avariety of such helper viruses for AAV are known in the art, includingadenoviruses, herpesviruses and poxviruses such as vaccinia. Theadenoviruses encompass a number of different subgroups, althoughAdenovirus type 5 of subgroup C is most commonly used. Numerousadenoviruses of human, non-human mammalian and avian origin are knownand available from depositories such as the ATCC. Viruses of the herpesfamily include, for example, herpes simplex viruses (HSV) andEpstein-Barr viruses (EBV), as well as cytomegaloviruses (CMV) andpseudorabies viruses (PRV); which are also available from depositoriessuch as ATCC.

“Helper virus function(s)” refers to function(s) encoded in a helpervirus genome which allow AAV replication and packaging (in conjunctionwith other requirements for replication and packaging described herein).As described herein, “helper virus function” may be provided in a numberof ways, including by providing helper virus or providing, for example,polynucleotide sequences encoding the requisite function(s) to aproducer cell in trans.

An “infectious” virus or viral particle is one that comprises apolynucleotide component which it is capable of delivering into a cellfor which the viral species is trophic. The term does not necessarilyimply any replication capacity of the virus. Assays for countinginfectious viral particles are described elsewhere in this disclosureand in the art. Viral infectivity can be expressed as the P:I ratio, orthe ratio of total viral particles to infective viral particles.

A “replication-competent” virus (e.g. a replication-competent AAV)refers to a phenotypically wild-type virus that is infectious, and isalso capable of being replicated in an infected cell (i.e. in thepresence of a helper virus or helper virus functions). In the case ofAAV, replication competence generally requires the presence offunctional AAV packaging genes. In general, rAAV vectors as describedherein are replication-incompetent in mammalian cells (especially inhuman cells) by virtue of the lack of one or more AAV packaging genes.Typically, such rAAV vectors lack any AAV packaging gene sequences inorder to minimize the possibility that replication competent AAV aregenerated by recombination between AAV packaging genes and an incomingrAAV vector. In many embodiments, rAAV vector preparations as describedherein are those which contain few if any replication competent AAV(rcAAV, also referred to as RCA) (e.g., less than about 1 rcAAV per 10²rAAV particles, less than about 1 rcAAV per 10⁴ rAAV particles, lessthan about 1 rcAAV per 10⁸ rAAV particles, less than about 1 rcAAV per10¹² rAAV particles, or no rcAAV).

The term “polynucleotide” refers to a polymeric form of nucleotides ofany length, including deoxyribonucleotides or ribonucleotides, oranalogs thereof. A polynucleotide may comprise modified nucleotides,such as methylated nucleotides and nucleotide analogs, and may beinterrupted by non-nucleotide components. If present, modifications tothe nucleotide structure may be imparted before or after assembly of thepolymer. The term polynucleotide, as used herein, refers interchangeablyto double- and single-stranded molecules. Unless otherwise specified orrequired, any embodiment of the invention described herein that is apolynucleotide encompasses both the double-stranded form and each of twocomplementary single-stranded forms known or predicted to make up thedouble-stranded form.

Nucleic acid hybridization reactions can be performed under conditionsof different “stringency”. Conditions that increase stringency of ahybridization reaction of widely known and published in the art. See,e.g., Sambrook et al. Molecular Cloning, A Laboratory Manual, 2nd Ed.,Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989,herein incorporated by reference. For example, see page 7.52 of Sambrooket al. Examples of relevant conditions include (in order of increasingstringency): incubation temperatures of 25° C., 37° C., 50° C. and 68°C.; buffer concentrations of 10×SSC, 6×SSC, 1×SSC, 0.1×SSC (where 1×SSCis 0.15 M NaCl and 15 mM citrate buffer) and their equivalents usingother buffer systems; formamide concentrations of 0%, 25%, 50%, and 75%;incubation times from 5 minutes to 24 hours; 1, 2, or more washingsteps; wash incubation times of 1, 2, or 15 minutes; and wash solutionsof 6×SSC, 1×SSC, 0.1×SSC, or deionized water. An example of stringenthybridization conditions is hybridization at 50° C. or higher and0.1×SSC (15 mM sodium chloride/1.5 mM sodium citrate). Another exampleof stringent hybridization conditions is overnight incubation at 42° C.in a solution: 50% formamide, 1×SSC (150 mM NaCl, 15 mM sodium citrate),50 mM sodium phosphate (pH 7.6), 5×Denhardt's solution, 10% dextransulfate, and 20 μg/ml denatured, sheared salmon sperm DNA, followed bywashing the filters in 0.1×SSC at about 65° C. As another example,stringent hybridization conditions comprise: prehybridization for 8hours to overnight at 65° C. in a solution comprising 6× single strengthcitrate (SSC) (1× SSC is 0.15 M NaCl, 0.015 M Na citrate; pH 7.0), 5×Denhardt's solution, 0.05% sodium pyrophosphate and 100 μg/ml herringsperm DNA; hybridization for 18-20 hours at 65° C. in a solutioncontaining 6× SSC, 1× Denhardt's solution, 100 μg/ml yeast tRNA and0.05% sodium pyrophosphate; and washing of filters at 65° C. for 1 h ina solution containing 0.2× SSC and 0.1% SDS (sodium dodecyl sulfate).

Stringent hybridization conditions are hybridization conditions that areat least as stringent as the above representative conditions. Otherstringent hybridization conditions are known in the art and may also beemployed to identify nucleic acids of this particular embodiment of theinvention.

“T_(m)” is the temperature in degrees Celsius at which 50% of apolynucleotide duplex made of complementary strands hydrogen bonded inanti-parallel direction by Watson-Crick base pairing dissociates intosingle strands under conditions of the experiment. T_(m) may bepredicted according to a standard formula, such as:

T _(m)=81.5+16.6 log[X ⁺]+0.41 (% G/C)−0.61 (% F)−600/L

where [X⁺] is the cation concentration (usually sodium ion, Na⁺) inmol/L; (% G/C) is the number of G and C residues as a percentage oftotal residues in the duplex; (% F) is the percent formamide in solution(wt/vol); and L is the number of nucleotides in each strand of theduplex.

A polynucleotide or polypeptide has a certain percent “sequenceidentity” to another polynucleotide or polypeptide, meaning that, whenaligned, that percentage of bases or amino acids are the same whencomparing the two sequences. Sequence similarity can be determined in anumber of different manners. To determine sequence identity, sequencescan be aligned using the methods and computer programs, including BLAST,available over the world wide web at ncbi.nlm nih.gov/BLAST/. Anotheralignment algorithm is FASTA, available in the Genetics Computing Group(GCG) package, from Madison, Wisconsin, USA, a wholly owned subsidiaryof Oxford Molecular Group, Inc. Other techniques for alignment aredescribed in Methods in Enzymology, vol. 266: Computer Methods forMacromolecular Sequence Analysis (1996), ed. Doolittle, Academic Press,Inc., a division of Harcourt Brace & Co., San Diego, Calif., USA. Ofparticular interest are alignment programs that permit gaps in thesequence. The Smith-Waterman is one type of algorithm that permits gapsin sequence alignments. See Meth. Mol. Biol. 70: 173-187 (1997). Also,the GAP program using the Needleman and Wunsch alignment method can beutilized to align sequences. See J. Mol. Biol. 48: 443-453 (1970)

Of interest is the BestFit program using the local homology algorithm ofSmith Waterman (Advances in Applied Mathematics 2: 482-489 (1981) todetermine sequence identity. The gap generation penalty will generallyrange from 1 to 5, usually 2 to 4 and in many embodiments will be 3. Thegap extension penalty will generally range from about 0.01 to 0.20 andin many instances will be 0.10. The program has default parametersdetermined by the sequences inputted to be compared. Preferably, thesequence identity is determined using the default parameters determinedby the program. This program is available also from Genetics ComputingGroup (GCG) package, from Madison, Wis., USA.

Another program of interest is the FastDB algorithm. FastDB is describedin Current Methods in Sequence Comparison and Analysis, MacromoleculeSequencing and Synthesis, Selected Methods and Applications, pp.127-149, 1988, Alan R. Liss, Inc. Percent sequence identity iscalculated by FastDB based upon the following parameters:

Mismatch Penalty: 1.00;

Gap Penalty: 1.00;

Gap Size Penalty: 0.33; and

Joining Penalty: 30.0.

A “gene” refers to a polynucleotide containing at least one open readingframe that is capable of encoding a particular protein after beingtranscribed and translated.

A “small interfering” or “short interfering RNA” or siRNA is a RNAduplex of nucleotides that is targeted to a gene interest (a “targetgene”). An “RNA duplex” refers to the structure formed by thecomplementary pairing between two regions of a RNA molecule. siRNA is“targeted” to a gene in that the nucleotide sequence of the duplexportion of the siRNA is complementary to a nucleotide sequence of thetargeted gene. In some embodiments, the length of the duplex of siRNAsis less than 30 nucleotides. In some embodiments, the duplex can be 29,28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11or 10 nucleotides in length. In some embodiments, the length of theduplex is 19-25 nucleotides in length. The RNA duplex portion of thesiRNA can be part of a hairpin structure. In addition to the duplexportion, the hairpin structure may contain a loop portion positionedbetween the two sequences that form the duplex. The loop can vary inlength. In some embodiments the loop is 5, 6, 7, 8, 9, 10, 11, 12 or 13nucleotides in length. The hairpin structure can also contain 3′ or 5′overhang portions. In some embodiments, the overhang is a 3′ or a 5′overhang 0, 1, 2, 3, 4 or 5 nucleotides in length.

“Recombinant,” as applied to a polynucleotide means that thepolynucleotide is the product of various combinations of cloning,restriction or ligation steps, and other procedures that result in aconstruct that is distinct from a polynucleotide found in nature. Arecombinant virus is a viral particle comprising a recombinantpolynucleotide. The terms respectively include replicates of theoriginal polynucleotide construct and progeny of the original virusconstruct.

A “control element” or “control sequence” is a nucleotide sequenceinvolved in an interaction of molecules that contributes to thefunctional regulation of a polynucleotide, including replication,duplication, transcription, splicing, translation, or degradation of thepolynucleotide. The regulation may affect the frequency, speed, orspecificity of the process, and may be enhancing or inhibitory innature. Control elements known in the art include, for example,transcriptional regulatory sequences such as promoters and enhancers. Apromoter is a DNA region capable under certain conditions of binding RNApolymerase and initiating transcription of a coding region usuallylocated downstream (in the 3′ direction) from the promoter.

“Operatively linked” or “operably linked” refers to a juxtaposition ofgenetic elements, wherein the elements are in a relationship permittingthem to operate in the expected manner For instance, a promoter isoperatively linked to a coding region if the promoter helps initiatetranscription of the coding sequence. There may be intervening residuesbetween the promoter and coding region so long as this functionalrelationship is maintained.

An “expression vector” is a vector comprising a region which encodes apolypeptide of interest, and is used for effecting the expression of theprotein in an intended target cell. An expression vector also comprisescontrol elements operatively linked to the encoding region to facilitateexpression of the protein in the target. The combination of controlelements and a gene or genes to which they are operably linked forexpression is sometimes referred to as an “expression cassette,” a largenumber of which are known and available in the art or can be readilyconstructed from components that are available in the art.

“Heterologous” means derived from a genotypically distinct entity fromthat of the rest of the entity to which it is being compared. Forexample, a polynucleotide introduced by genetic engineering techniquesinto a plasmid or vector derived from a different species is aheterologous polynucleotide. A promoter removed from its native codingsequence and operatively linked to a coding sequence with which it isnot naturally found linked is a heterologous promoter. Thus, forexample, an rAAV that includes a heterologous nucleic acid encoding aheterologous gene product is an rAAV that includes a nucleic acid notnormally included in a naturally-occurring, wild-type AAV, and theencoded heterologous gene product is a gene product not normally encodedby a naturally-occurring, wild-type AAV.

The terms “genetic alteration” and “genetic modification” (andgrammatical variants thereof), are used interchangeably herein to referto a process wherein a genetic element (e.g., a polynucleotide) isintroduced into a cell other than by mitosis or meiosis. The element maybe heterologous to the cell, or it may be an additional copy or improvedversion of an element already present in the cell. Genetic alterationmay be effected, for example, by transfecting a cell with a recombinantplasmid or other polynucleotide through any process known in the art,such as electroporation, calcium phosphate precipitation, or contactingwith a polynucleotide-liposome complex. Genetic alteration may also beeffected, for example, by transduction or infection with a DNA or RNAvirus or viral vector. Generally, the genetic element is introduced intoa chromosome or mini-chromosome in the cell; but any alteration thatchanges the phenotype and/or genotype of the cell and its progeny isincluded in this term.

A cell is said to be “stably” altered, transduced, genetically modified,or transformed with a genetic sequence if the sequence is available toperform its function during extended culture of the cell in vitro.Generally, such a cell is “heritably” altered (genetically modified) inthat a genetic alteration is introduced which is also inheritable byprogeny of the altered cell.

The terms “polypeptide,” “peptide,” and “protein” are usedinterchangeably herein to refer to polymers of amino acids of anylength. The terms also encompass an amino acid polymer that has beenmodified; for example, disulfide bond formation, glycosylation,lipidation, phosphorylation, or conjugation with a labeling component.Polypeptides such as “CFTR,” “p53,” “EPO” and the like, when discussedin the context of delivering a gene product to a mammalian subject, andcompositions therefor, refer to the respective intact polypeptide, orany fragment or genetically engineered derivative thereof, that retainsthe desired biochemical function of the intact protein. Similarly,references to CFTR, p53, EPO genes, and other such genes for use indelivery of a gene product to a mammalian subject (which may be referredto as “transgenes” to be delivered to a recipient cell), includepolynucleotides encoding the intact polypeptide or any fragment orgenetically engineered derivative possessing the desired biochemicalfunction.

An “isolated” plasmid, nucleic acid, vector, virus, host cell, or othersubstance refers to a preparation of the substance devoid of at leastsome of the other components that may also be present where thesubstance or a similar substance naturally occurs or is initiallyprepared from. Thus, for example, an isolated substance may be preparedby using a purification technique to enrich it from a source mixture.Enrichment can be measured on an absolute basis, such as weight pervolume of solution, or it can be measured in relation to a second,potentially interfering substance present in the source mixture.Increasing enrichments of the embodiments of this invention areincreasingly more isolated. An isolated plasmid, nucleic acid, vector,virus, host cell, or other substance is in some embodiments purified,e.g., from about 80% to about 90% pure, at least about 90% pure, atleast about 95% pure, at least about 98% pure, or at least about 99%, ormore, pure.

As used herein, the terms “treatment,” “treating,” and the like, referto obtaining a desired pharmacologic and/or physiologic effect. Theeffect may be prophylactic in terms of completely or partiallypreventing a disease or symptom thereof and/or may be therapeutic interms of a partial or complete cure for a disease and/or adverse affectattributable to the disease. “Treatment,” as used herein, covers anytreatment of a disease in a mammal, particularly in a human, andincludes: (a) preventing the disease from occurring in a subject whichmay be predisposed to the disease or at risk of acquiring the diseasebut has not yet been diagnosed as having it; (b) inhibiting the disease,i.e., arresting its development; and (c) relieving the disease, i.e.,causing regression of the disease.

The terms “individual,” “host,” “subject,” and “patient” are usedinterchangeably herein, and refer to a mammal, including, but notlimited to, human and non-human primates, including simians and humans;mammalian sport animals (e.g., horses); mammalian farm animals (e.g.,sheep, goats, etc.); mammalian pets (dogs, cats, etc.); and rodents(e.g., mice, rats, etc.).

Before the present invention is further described, it is to beunderstood that this invention is not limited to particular embodimentsdescribed, as such may, of course, vary. It is also to be understoodthat the terminology used herein is for the purpose of describingparticular embodiments only, and is not intended to be limiting, sincethe scope of the present invention will be limited only by the appendedclaims.

Where a range of values is provided, it is understood that eachintervening value, to the tenth of the unit of the lower limit unlessthe context clearly dictates otherwise, between the upper and lowerlimit of that range and any other stated or intervening value in thatstated range, is encompassed within the invention. The upper and lowerlimits of these smaller ranges may independently be included in thesmaller ranges, and are also encompassed within the invention, subjectto any specifically excluded limit in the stated range. Where the statedrange includes one or both of the limits, ranges excluding either orboth of those included limits are also included in the invention.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein can also beused in the practice or testing of the present invention, the preferredmethods and materials are now described. All publications mentionedherein are incorporated herein by reference to disclose and describe themethods and/or materials in connection with which the publications arecited.

It must be noted that as used herein and in the appended claims, thesingular forms “a,” “an,” and “the” include plural referents unless thecontext clearly dictates otherwise. Thus, for example, reference to “anrAAV vector” includes a plurality of such vectors and reference to “themutant AAV capsid protein” includes reference to one or more mutant AAVcapsid proteins and equivalents thereof known to those skilled in theart, and so forth.

The publications discussed herein are provided solely for theirdisclosure prior to the filing date of the present application. Nothingherein is to be construed as an admission that the present invention isnot entitled to antedate such publication by virtue of prior invention.Further, the dates of publication provided may be different from theactual publication dates which may need to be independently confirmed.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides mutant adeno-associated virus (AAV) thatexhibit altered capsid properties, e.g., reduced binding to neutralizingantibodies in serum and/or altered heparin binding and/or alteredinfectivity of particular cell types. The present invention furtherprovides libraries of mutant AAV comprising one or more mutations in acapsid gene. The present invention further provides methods ofgenerating the mutant AAV, mutant AAV libraries, and compositionscomprising the mutant AAV or mutant AAV libraries. The present inventionfurther provides recombinant AAV (rAAV) virions that comprise a mutantcapsid protein. The present invention further provides nucleic acidscomprising nucleotide sequences that encode mutant capsid proteins, andhost cells comprising the nucleic acids. The present invention furtherprovides methods of delivering a gene product to an individual, themethods generally involving administering an effective amount of asubject rAAV virion to an individual in need thereof. In manyembodiments, a subject mutant AAV virion, a subject nucleic acid, etc.,is isolated.

A subject mutant AAV virion or a subject rAAV virion exhibits one ormore of the following properties: 1) increased heparan sulfate bindingaffinity relative to wild-type AAV; 2) decreased heparan sulfate bindingaffinity relative to wild-type AAV; 3) increased infectivity of a cellthat is resistant to infection with AAV, or that is less permissive toinfection with AAV than a prototypical permissive cell; 4) increasedevasion of neutralizing antibodies; and 5) increased ability to cross anendothelial cell layer.

A subject nucleic acid encoding a mutant AAV capsid protein is usefulfor generating recombinant AAV virions that exhibit altered propertiessuch as increased heparan sulfate binding, decreased heparan sulfatebinding, increased infectivity of a cell that is resistant to infectionwith AAV or that is less permissive to infection with AAV, increasedevasion of neutralizing antibodies, increased ability to cross anendothelial cell layer, and the like. A subject rAAV virion is usefulfor delivering a gene product to an individual.

Cell membrane-associated heparan sulfate proteoglycan is a primary cellsurface receptor for AAV, e.g., AAV-2. Increased heparan sulfate binding(e.g., increased heparin affinity) is advantageous where, e.g., the rAAVparticle is being delivered in a localized manner, e.g., where diffusionof the rAAV particle away from the site of delivery is not desired. Manycell types produce heparan sulfate, which remains on the surface of thecell or in the immediate environment of the cell. Thus, an rAAV virionwith increased heparan sulfate binding affinity would remain relativelyclose to the site of administration. For example, localized delivery ofa subject rAAV virion is advantageous for delivery of a gene product toa tumor that is localized to a particular anatomical site (but not,e.g., to surrounding non-cancerous tissue), for delivery of a geneproduct to a diseased cardiac vessel (but not to the surrounding healthyheart tissue), etc.

In many embodiments of the present invention, AAV-2, and mutants ofAAV-2, are exemplified. However, the exemplification of AAV-2 herein isin no way meant to be limiting. Those skilled in the art can readilyadapt the methods as discussed herein to generate capsid mutants ofother AAV, including, e.g., AAV-3, AAV-4, AAV-5, etc. Thus, e.g., wherean AAV binds to the β₅ subunit of integrin α_(v)β₅, the presentinvention contemplates mutant AAV that exhibit increased or decreasedbinding to the β₅ subunit of integrin α_(v)β₅, compared to thecorresponding wild-type AAV. As another example, where an AAV (e.g.,AAV-4) binds to O-linked sialic acid, the present invention contemplatesmutant AAV that exhibit increased or decreased binding to O-linkedsialic acid. As another example, where an AAV (e.g., AAV-5) binds toN-linked sialic acid or to a platelet-derived growth factor receptor(PDGFR), the present invention contemplates mutant AAV that exhibitincreased or decreased binding to N-linked sialic acid or PDGFR. See,e.g., Kaludov et al. ((2001) J. Virol. 75:6884); Pasquale et al. ((2003)Nat. Med. 9:1306); and Walters et al. ((2001) J. Biol. Chem. 276:20610)for descriptions of AAV receptors.

Increased heparin affinity is also advantageous in that it confersincreased infectivity of cell types that are typically refractory toinfection with AAV, e.g., non-permissive cell types and“less-permissive” cell types (e.g., cells that are less permissive thana prototypical permissive cell). Such cell types include those withrelatively low amounts of heparan sulfate on their surface. Increasedheparan sulfate binding affinity allows an increased level of binding tocells that have relatively low levels of surface heparan sulfate, andtherefore leads to increased infectivity of these cells. An example ofcells that are refractory to infection with AAV is a stem cell. Thus, asubject rAAV virion is advantageous because it can infect stem cells andcan deliver gene products to stem cells. Other examples of cells thatare non-permissive or less permissive to infection with AAV include lungepithelial cells and hepatocytes.

Decreased heparan sulfate binding (e.g., decreased heparin affinity) isadvantageous for therapeutic strategies in which more widespread, orsystemic delivery of a subject rAAV virion is desired. Such rAAV virionsdiffuse away from the site of administration, and thus infect a greaternumber of cells than rAAV virions with wild-type capsid protein(s).

Decreased binding to neutralizing antibodies is advantageous.Neutralizing antibodies bind to wild-type capsid proteins. Binding ofneutralizing antibodies to wild-type capsid proteins may have severaleffects, including limiting the residence time of an rAAV virions thatcomprises wild-type capsid proteins in the viral particle, preventingthe virus from binding to the cell surface, aggregating the virus,induction of structural alterations in the capsid, and prevention ofviral disassembly and uncoating (a step necessary to release the DNA).An rAAV particle that has decreased binding to neutralizing antibodiesthus has increased capacity to infect cells, and increased residencetime in the body of an individual administered with the rAAV virion.Thus, the effective duration of delivery of gene product is increased.

Increased ability to cross an endothelial cell layer allows the rAAVvirion to gain access to tissues and cells that are separated from thesite of administration by an endothelial cell layer. For example, theblood-brain barrier, the tumor vasculature, and the cardiovascularsystem all present endothelial cell layers that form a barrier to accessof a particular anatomical site. A subject rAAV virion thus may exhibitone or more of the following properties: 1) increased ability to crossthe blood-brain barrier; 2) increased ability to cross the tumorvasculature and infect tumor cells; and 3) increased ability to crossthe endothelial layer within the heart.

Mutant Adeno-Associated Virus Virions

The present invention provides mutant adeno-associated virus comprisingmutant capsid proteins that exhibit altered capsid properties. By virtueof comprising one or more mutant capsid proteins, a subject mutant AAVexhibits one or more of the following properties: 1) increased heparinbinding affinity relative to wild-type AAV; 2) decreased heparin bindingaffinity relative to wild-type AAV; 3) increased infectivity of a cellthat is resistant to infection with AAV; 4) increased evasion ofneutralizing antibodies; and 5) increased ability to cross anendothelial cell layer. The properties of a subject mutant AAV arecompared to a corresponding parental, AAV, e.g., a wild-type AAV. Insome embodiments, the corresponding parental AAV is a chimeric AAV,e.g., an AAV with a chimeric capsid protein comprising a first stretchof contiguous amino acids from a first AAV serotype and a second stretchof contiguous amino acids from a second AAV serotype. In someembodiments, the corresponding parental AAV is wild-type AAV. Thus,e.g., where the parental, wild-type AAV is AAV-2, and the subject mutantAAV is a mutant of wild-type AAV-2, the properties of the subject mutantare compared to that same property of wild-type AAV-2.

Mutants With Increased Heparin Affinity

In some embodiments, a capsid protein encoded by a subject mutant AAVexhibits increased binding affinity to heparan sulfate relative towild-type AAV. In these embodiments, a capsid protein encoded by asubject mutant AAV exhibits at least about 10%, at least about 15%, atleast about 20%, at least about 25%, at least about 30%, at least about35%, at least about 40%, at least about 45%, at least about 50%, atleast about 55%, at least about 60%, at least about 65%, at least about70%, at least about 75%, at least about 80%, at least about 85%, atleast about 90%, at least about 95%, at least about 2-fold, at least2.5-fold, at least about 5-fold, at least about 10-fold, at least about15-fold, at least about 50 fold, at least about 75-fold, or at leastabout 100-fold or more, higher affinity for heparan sulfate thanwild-type AAV capsid. Because heparin is a molecule that is structurallysimilar to heparan sulfate, heparin is frequently used to determineexperimentally whether a capsid protein has altered binding to heparansulfate. Thus, the terms “heparin binding affinity,” and “heparansulfate binding affinity,” and similar terms, are used interchangeablyherein.

For example, whereas the binding affinity of AAV-2 to heparin has a K(d)value of approximately 2.0 nM, a subject mutant AAV has a bindingaffinity to heparin that is at least about 10%, at least about 15%, atleast about 20%, at least about 25%, at least about 30%, at least about35%, at least about 40%, at least about 45%, at least about 50%, atleast about 55%, at least about 60%, at least about 65%, at least about70%, at least about 75%, at least about 80%, at least about 85%, atleast about 90%, at least about 95%, at least about 2-fold, at least2.5-fold, at least about 5-fold, at least about 10-fold, at least about15-fold, at least about 50 fold, at least about 75-fold, or at leastabout 100-fold or more, higher than the affinity of wild-type AAV-2 toheparin.

Typically, wild-type AAV elutes from a heparin affinity chromatographymedium with a NaCl concentration in a range of from about 450 mM toabout 550 mM. In some embodiments, a subject mutant AAV elutes from aheparin affinity chromatography medium with a NaCl concentration ofgreater than about 550 mM, e.g., from about 575 mM NaCl to about 600 mMNaCl, from about 600 mM NaCl to about 625 mM NaCl, from about 625 mMNaCl to about 650 mM NaCl, from about 650 mM NaCl to about 675 mM NaCl,from about 675 mM NaCl to about 700 mM NaCl, from about 700 mM NaCl toabout 725 mM NaCl, from about 725 mM NaCl to about 750 mM NaCl, fromabout 750 mM NaCl to about 775 mM NaCl, or from about 775 mM NaCl toabout 800 mM NaCl, or higher.

Mutants With Decreased Heparin Affinity

In other embodiments, a subject mutant AAV exhibits a lower affinity forheparan sulfate than wild-type AAV. In these embodiments, a subjectmutant AAV, when packaged in a viral particle, has at least about 10%,at least about 15%, at least about 20%, at least about 25%, at leastabout 30%, at least about 35%, at least about 40%, at least about 45%,at least about 50%, at least about 55%, at least about 60%, at leastabout 65%, at least about 70%, at least about 75%, at least about 80%,or at least about 85% lower affinity for heparin than wild-type AAV(e.g., wild-type AAV-2). In some embodiments, a subject mutant AAV, whenpackaged into a viral particle, elutes from a heparin affinitychromatography medium with concentration of NaCl in the range of fromabout 440 mM NaCl to about 400 mM NaCl, from about 400 mM NaCl to about375 mM NaCl, from about 375 mM NaCl to about 350 mM NaCl, from about 350mM NaCl to about 325 mM NaCl, from about 325 mM NaCl to about 300 mMNaCl, from about 300 mM NaCl to about 275 mM NaCl, from about 275 mMNaCl to about 250 mM NaCl, from about 250 mM NaCl to about 225 mM NaCl,from about 225 mM NaCl to about 200 mM NaCl or lower.

Heparin binding affinity can be determined using any known assay. Forexample, affinity of variant capsids for heparan sulfate can be measuredby binding viral particles to immobilized heparin. See, e.g., Qui et al.(2000) Virology 269:137-147.

Neutralizing Antibody-Evading Mutants

In some embodiments, a subject mutant AAV exhibits increased resistanceto neutralizing antibodies compared to wild-type AAV (“wt AAV”) or AAVcomprising a wild-type capsid protein. In these embodiments, a subjectmutant AAV has from about 10-fold to about 10,000-fold greaterresistance to neutralizing antibodies than wt AAV, e.g., a subjectmutant AAV has from about 10-fold to about 25-fold, from about 25-foldto about 50-fold, from about 50-fold to about 75-fold, from about75-fold to about 100-fold, from about 100-fold to about 150-fold, fromabout 150-fold to about 200-fold, from about 200-fold to about 250-fold,from about 250-fold to about 300-fold, at least about 350-fold, at leastabout 400-fold, from about 400-fold to about 450-fold, from about450-fold to about 500-fold, from about 500-fold to about 550-fold, fromabout 550-fold to about 600-fold, from about 600-fold to about 700-fold,from about 700-fold to about 800-fold, from about 800-fold to about900-fold, from about 900-fold to about 1000-fold, from about 1,000-foldto about 2,000-fold, from about 2,000-fold to about 3,000-fold, fromabout 3,000-fold to about 4,000-fold, from about 4,000-fold to about5,000-fold, from about 5,000-fold to about 6,000-fold, from about6,000-fold to about 7,000-fold, from about 7,000-fold to about8,000-fold, from about 8,000-fold to about 9,000-fold, or from about9,000-fold to about 10,000-fold greater resistance to neutralizingantibodies than a wild-type AAV or an AAV comprising a wild-type capsidprotein.

In some embodiments, a subject mutant AAV exhibits decreased binding toa neutralizing antibody that binds a wild-type AAV capsid protein. Forexample, a subject mutant AAV exhibits from about 10-fold to about10,000-fold reduced binding to a neutralizing antibody that binds awild-type AAV capsid protein. For example, a subject mutant AAV exhibitsfrom about 10-fold to about 25-fold, from about 25-fold to about50-fold, from about 50-fold to about 75-fold, from about 75-fold toabout 100-fold, from about 100-fold to about 150-fold, from about150-fold to about 200-fold, from about 200-fold to about 250-fold, fromabout 250-fold to about 300-fold, at least about 350-fold, at leastabout 400-fold, from about 400-fold to about 450-fold, from about450-fold to about 500-fold, from about 500-fold to about 550-fold, fromabout 550-fold to about 600-fold, from about 600-fold to about 700-fold,from about 700-fold to about 800-fold, from about 800-fold to about900-fold, from about 900-fold to about 1000-fold, from about 1,000-foldto about 2,000-fold, from about 2,000-fold to about 3,000-fold, fromabout 3,000-fold to about 4,000-fold, from about 4,000-fold to about5,000-fold, from about 5,000-fold to about 6,000-fold, from about6,000-fold to about 7,000-fold, from about 7,000-fold to about8,000-fold, from about 8,000-fold to about 9,000-fold, or from about9,000-fold to about 10,000-fold reduced binding (e.g., reduced affinity)to a neutralizing antibody that binds a wild-type capsid AAV protein,compared to the binding affinity of the antibody to wild-type AAV capsidprotein.

In some embodiments, an anti-AAV neutralizing antibody binds to asubject neutralizing antibody escape mutant AAV with an affinity of lessthan about 10⁻⁷ M, less than about 5×10⁻⁶ M, less than about 10⁻⁶ M,less than about 5×10⁻⁵ M, less than about 10⁻⁵ M, less than about 10⁻⁴M, or lower.

In some embodiments, a subject mutant AAV exhibits increased in vivoresidence time compared to a wild-type AAV. For example, a subjectmutant AAV exhibits a residence time that is at least about 10%, atleast about 25%, at least about 50%, at least about 100%, at least about3-fold, at least about 5-fold, at least about 10-fold, at least about25-fold, at least about 50-fold, at least about 100-fold, or more,longer than the residence time of a wild-type AAV.

Whether a given mutant AAV exhibits reduced binding to a neutralizingantibody and/or increased resistance to neutralizing antibody can bedetermined using any known assay, including the assay described inExample 1. For example, mutant AAV is contacted with a permissive celltype, e.g., 293 cells, in the presence of neutralizing antibody. Acontrol sample contains the cells, mutant AAV, and no neutralizingantibody. After a suitable time, the cells are contacted withadenovirus, and AAV particles are detected. The level of AAV particlesis compared to the amount of AAV particles that are generated in theabsence of neutralizing antibody.

Mutants With Increased Infectivity of a Non-Permissive Cell

In some embodiments, a subject mutant AAV virion that exhibits increasedinfectivity of cells that are non-permissive to infection with AAV, andcells that are less permissive to infection with AAV. A subject mutantAAV virion comprises a variant AAV capsid protein, where the variant AAVcapsid protein comprises at least one amino acid substitution relativeto a corresponding parental AAV capsid protein, and where the variantcapsid protein confers increased infectivity of a non-permissive cellcompared to the infectivity of the non-permissive cell by an AAV virioncomprising the corresponding parental AAV capsid protein. Thus, asubject mutant AAV virion exhibits increased infectivity of anonpermissive cell compared to a corresponding parental AAV virioncomprising a corresponding parental AAV capsid protein.

Cells that are non-permissive to infection with AAV, and cells that areless permissive to infection with AAV, are collectively referred toherein as “non-permissive cells.” When a population of permissive cellsis contacted in vitro or in vivo with AAV at a multiplicity of infection(moi) of 5, from about 70% to about 100% of the cell population becomesinfected with the AAV. When a population of non-permissive cells iscontacted in vitro or in vivo with AAV at an moi of 5, less than about70% of the population becomes infected with AAV, e.g., no greater thanfrom about 60% to about 69%, from about 50% to about 60%, from about 40%to about 50%, from about 30% to about 40%, from about 20% to about 30%,from about 10% to about 20%, or from about 1% to about 10%, of thepopulation becomes infected with AAV, and in some cell types,essentially none of the cells becomes infected with AAV.

Whether a cell is permissive or non-permissive to infection with AAV canbe readily determined by contacting in vitro or in vivo a population ofa particular cell type with an rAAV construct that comprises anucleotide sequence encoding a protein that provides a detectable signal(e.g., a fluorescent protein such as a green fluorescent protein), at anmoi of 5. The proportion of cells that become positive for thedetectable protein is an indication of the percentage of cells thatbecame infected with the rAAV. Where from about 0% to about 69% of thecells become infected with the rAAV, the cells are said to benon-permissive to infection with AAV. Where from about 70% to about 100%of the cells become infected with the rAAV, the cells are said to bepermissive to infection with AAV. Infectivity can be expressed relativeto infectivity of 293 cells. In some embodiments, a non-permissive cellexhibits reduced infectivity with AAV compared to 293 cells, e.g., anon-permissive cell exhibits less than about 70% of the infectivity of293 cells to AAV, e.g., a non-permissive cells exhibits less than about70%, less than about 60%, less than about 50%, less than about 40%, lessthan about 30%, less than about 20%, less than about 10%, or less, ofthe infectivity of 293 cells to AAV.

In some embodiments, a subject mutant AAV exhibits increased ability toinfect a cell that is relatively refractory to AAV infection (e.g., anon-permissive cell). In these embodiments, a subject mutant AAVexhibits at least about 10%, at least about 20%, at least about 30%, atleast about 40%, at least about 50%, at least about 60%, at least about70%, at least about 80%, at least about 90%, at least about 2-fold, atleast about 4-fold, at least about 5-fold, at least about 10-fold, atleast about 25-fold, or more, greater infectivity of a non-permissivecell than a wild-type AAV.

Examples of cells that are relatively refractory to AAV infectioninclude stem cells. Further examples of non-permissive cell typesinclude, but are not limited to, lung epithelial cells, and hepatocytes.

The term “stem cell” is used herein to refer to a mammalian cell thathas the ability both to self-renew, and to generate differentiatedprogeny (see, e.g., Morrison et al. (1997) Cell 88:287-298). Generally,stem cells also have one or more of the following properties: an abilityto undergo asynchronous, or symmetric replication, that is where the twodaughter cells after division can have different phenotypes; extensiveself-renewal capacity; capacity for existence in a mitotically quiescentform; and clonal regeneration of all the tissue in which they exist, forexample the ability of hematopoietic stem cells to reconstitute allhematopoietic lineages. “Progenitor cells” differ from stem cells inthat they typically do not have the extensive self-renewal capacity, andoften can only regenerate a subset of the lineages in the tissue fromwhich they derive, for example only lymphoid, or erythroid lineages in ahematopoietic setting.

Stem cells may be characterized by both the presence of markersassociated with specific epitopes identified by antibodies and theabsence of certain markers as identified by the lack of binding ofspecific antibodies. Stem cells may also be identified by functionalassays both in vitro and in vivo, particularly assays relating to theability of stem cells to give rise to multiple differentiated progeny.

Stem cells of interest include hematopoietic stem cells and progenitorcells derived therefrom (U.S. Pat. No. 5,061,620); neural crest stemcells (see Morrison et al. (1999) Cell 96:737-749); adult neural stemcells and neural progenitor cells; embryonic stem cells; mesenchymalstem cells; mesodermal stem cells; etc. Other hematopoietic “progenitor”cells of interest include cells dedicated to lymphoid lineages, e.g.immature T cell and B cell populations.

Structural Features

A subject mutant AAV virion comprises a mutation in at least one capsidprotein (e.g., at least one of VP1, VP2, and VP3). Thus, at least one ofVP1, VP2, and VP3 has at least one amino acid substitution compared to acorresponding parental AAV capsid protein, e.g., a wild-type AAV capsidprotein. In some embodiments, at least one of VP1, VP2, and VP3 has fromone to about 50 amino acid substitutions compared to wild-type AAV VP1,VP2, and VP3, e.g., from about one to about 5, from about 5 to about 10,from about 10 to about 15, from about 15 to about 20, from about 20 toabout 25, from about 25 to about 30, from about 30 to about 40, fromabout 40 to about 45, or from about 45 to about 50, amino acidsubstitutions compared to wild-type AAV VP1, VP2, and VP3.Alternatively, a subject mutant AAV virion comprises one or more aminoacid deletions and/or insertions in at least one capsid protein relativeto a corresponding parental AAV capsid protein, e.g., a wild-type capsidprotein. In some embodiments, a subject mutant AAV virion comprises oneor more amino acid substitutions and/or deletions and/or insertions in acapsid protein relative to a corresponding parental AAV capsid protein,e.g., a wild-type capsid protein. In some embodiments, a subject mutantAAV virion comprises one or more amino acid substitutions compared to acorresponding parental AAV capsid protein, and can further comprise fromone to about 10 amino acid deletions compared to a correspondingparental AAV capsid protein. In some embodiments, a subject mutant AAVvirion comprises one or more amino acid substitutions compared to acorresponding parental AAV capsid protein, and does not comprise anyamino acid insertions, e.g., does not comprise any insertions of aminoacids that provide an epitope not present in a corresponding parentalAAV capsid protein (e.g., a wild-type AAV capsid protein).

The corresponding, parental AAV capsid protein can be a wild-type capsidprotein (e.g., a wild-type AAV2 capsid protein, a wild-type AAVS capsidprotein, etc.). The corresponding, parental AAV capsid protein can be achimeric AAV capsid protein, e.g., an AAV capsid protein comprising afirst contiguous stretch of amino acids of a first AAV serotype, and asecond contiguous stretch of amino acids of a second AAV serotype. Asone non-limiting example, a parental AAV capsid protein can comprise acontiguous stretch of from about 10 amino acids to about 120 amino acidsof the amino-terminal 120 amino acids of AAV2; and a contiguous stretchof from about 400 amino acids to about 550 contiguous amino acids of thecarboxyl-terminal 550 amino acid of AAVS.

In some embodiments, a subject mutant AAV virion exhibits reducedbinding to neutralizing antibody compared to wild-type AAV, andcomprises a VP1 that has an amino acid sequence that has at least about85%, at least about 90%, at least about 95%, at least about 98%, or atleast about 99%, or greater, amino acid sequence identity to an aminoacid sequence as set forth in one of SEQ ID NOs:7, 9, 11, 13, 15, 17,19, 21, 23, 25, 27, 29, 31, and 33, or as set forth in FIGS. 5A-C orFIGS. 7A-D. In some embodiments, a subject mutant AAV virion exhibitsreduced binding to neutralizing antibody compared to wild-type AAV, andcomprises a VP1 that has an amino acid sequence that has from 1-5, from5-10, or from 10-20 amino acid differences from an amino acid sequenceas set forth in one of SEQ ID NOs:7, 9, 11, 13, 15, 17, 19, 21, 23, 25,27, 29, 31, and 33 or as set forth in FIGS. 5A-C or FIGS. 7A-D. In someembodiments, a subject mutant AAV virion exhibits reduced binding toneutralizing antibody compared to wild-type AAV, and comprises a VP1that has an amino acid sequence as set forth in one of SEQ ID NOs:7, 9,11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, and 33 or as set forth inFIGS. 5A-C or FIGS. 7A-D.

In some embodiments, a subject mutant AAV virion exhibits increasedheparan sulfate affinity compared to wild-type AAV, and comprises a VP1that has an amino acid sequence that has at least about 85%, at leastabout 90%, at least about 95%, at least about 98%, or at least about99%, or greater, amino acid sequence identity to an amino acid sequenceas set forth in SEQ ID NO:35 or SEQ ID NO:37, or as set forth in FIGS.9A-C (D14H1 or D14L3). In some embodiments, a subject mutant AAV virionexhibits increased heparan sulfate affinity compared to wild-type AAV,and comprises a VP1 that has an amino acid sequence that has from 1-5,from 5-10, or from 10-20 amino acid differences from an amino acidsequence as set forth in SEQ ID NO:35 or SEQ ID NO:37, or as set forthin FIGS. 9A-C (D14H1 or D14L3). In some embodiments, a subject mutantAAV virion exhibits increased heparan sulfate affinity compared towild-type AAV, and comprises a VP1 that has an amino acid sequence asset forth in SEQ ID NO:35 or SEQ ID NO:37, or as set forth in FIGS. 9A-C(D14H1 or D14L3).

In some embodiments, a subject mutant AAV virion exhibits reducedheparan sulfate affinity compared to wild-type AAV, and comprises a VP1that has an amino acid sequence that has at least about 85%, at leastabout 90%, at least about 95%, at least about 98%, or at least about99%, or greater, amino acid sequence identity to an amino acid sequenceas set forth in SEQ ID NO:39 or SEQ ID NO:41, or as set forth in FIGS.9A-C (P1BH1 or P1BH2). In some embodiments, a subject mutant AAV virionexhibits reduced heparan sulfate affinity compared to wild-type AAV, andcomprises a VP1 that has an amino acid sequence that has from 1-5, from5-10, or from 10-20 amino acid differences from an amino acid sequenceas set forth in SEQ ID NO:39 or SEQ ID NO:41, or as set forth in FIGS.9A-C (P1BH1 or P1BH2). In some embodiments, a subject mutant AAV virionexhibits decreased heparan sulfate affinity compared to wild-type AAV,and comprises a VP1 that has an amino acid sequence as set forth in SEQID NO:39 or SEQ ID NO:41, or as set forth in FIGS. 9A-C (P1BH1 orP1BH2).

In some embodiments, a subject mutant AAV virion exhibits increasedinfectivity of a non-permissive lung epithelial cell compared to acorresponding parental AAV (e.g., a wild-type AAV; or a chimeric AAV),and comprises a VP1 that has an amino acid sequence that has at leastabout 85%, at least about 90%, at least about 95%, at least about 98%,or at least about 99%, or greater, amino acid sequence identity to theamino acid sequence as set forth in SEQ ID NO:42, or as set forth inFIGS. 10A-B (AAV2.5T), where the VP1 of the mutant AAV virion comprisesan amino acid sequence that has from 1-5, from 5-10, from 10-20, from 20to 25, or from 25-45 amino acid differences from the amino acid sequenceas set forth in SEQ ID NO:43 (AAVS). In some embodiments, a subjectmutant AAV virion exhibits increased infectivity of a non-permissivelung epithelial cell compared to a corresponding parental AAV (e.g., awild-type AAV; or a chimeric AAV), and comprises a VP1 that comprises anA581T substitution compared to the amino acid sequence of an AAVS capsidprotein set forth in SEQ ID NO:43.

In some embodiments, a subject mutant AAV virion exhibits increasedinfectivity of a non-permissive stem cell compared to a correspondingparental AAV (e.g., a wild-type AAV), and comprises a VP1 that comprisesan amino acid sequence that has at least about 85%, at least about 90%,at least about 95%, at least about 98%, or at least about 99%, orgreater, amino acid sequence identity to the amino acid sequence as setforth in any one of SEQ ID NOs:44-58, where the VP1 of the mutant AAVvirion comprises from 1 to 5, from 5 to 10, or from 10 to 15 amino acidsubstitutions compared to the amino acid sequence of AAV2 as set forthin SEQ ID NO:5. In some embodiments, a subject mutant AAV virionexhibits increased infectivity of a non-permissive stem cell compared towild-type AAV2, and comprises an R459G substitution compared to theamino acid sequence of AAV2 as set forth in SEQ ID NO:5. In someembodiments, a subject mutant AAV virion exhibits increased infectivityof a non-permissive stem cell compared to wild-type AAV2, and comprisesa V7081 substitution and a P250S substitution compared to the amino acidsequence of AAV2 as set forth in SEQ ID NO:5.

In some embodiments, a subject mutant AAV virion exhibits increasedinfectivity of a non-permissive stem cell compared to a correspondingparental AAV (e.g., a wild-type AAV), and comprises a VP1 that comprisesan amino acid sequence that has at least about 85%, at least about 90%,at least about 95%, at least about 98%, or at least about 99%, orgreater, amino acid sequence identity to the amino acid sequence as setforth in SEQ ID NO: 59, where the VP1 of the mutant AAV virion comprisesfrom 1 to 5, from 5 to 10, or from 10 to 15 amino acid substitutionscompared to the amino acid sequence of AAV2 as set forth in SEQ ID NO:5.In some embodiments, a subject mutant AAV virion exhibits increasedinfectivity of a non-permissive stem cell compared to wild-type AAV2,and comprises an S85G substitution and an R459G substitution compared tothe amino acid sequence of AAV2 as set forth in SEQ ID NO:5.

In some embodiments, a subject mutant AAV virion comprises wild-typeRep78, Rep68, Rep52, and Rep40 proteins. In other embodiments, a subjectmutant AAV comprises, in addition to one or more mutant capsid proteins,one or more mutations in one or more of Rep78, Rep68, Rep52, and Rep40proteins.

Nucleic Acids and Host Cells

The present invention provides nucleic acids comprising nucleotidesequences encoding a mutant AAV capsid protein, as well as host cellscomprising a subject nucleic acid. The nucleic acids and host cells areuseful for generating rAAV virions, as described below. A subjectnucleic acid encodes one or more of VP1, VP2, and VP3 comprising one ormore amino acid substitutions. A subject nucleic acid comprises anucleotide sequence encoding at least one of VP1, VP2, and VP3, whereinthe encoded capsid protein comprises from one to about 15 amino acidsubstitutions compared to a wild-type AAV capsid protein, e.g., fromabout one to about 5, from about 5 to about 10, from about 10 to about15, from about 15 to about 20, or from about 20 to about 25 amino acidsubstitutions compared to a corresponding parental AAV capsid protein(e.g., compared to a wild-type AAV capsid protein). The encoded capsidprotein may, alternatively or in addition, comprise one or more aminoacid deletions and/or insertions relative to a wild-type AAV capsidprotein. In some embodiments, a the encoded mutant capsid proteincomprises one or more amino acid substitutions and/or deletions and/orinsertions relative to a corresponding parental AAV capsid protein,e.g., a wild-type capsid protein. In some embodiments, the encodedmutant capsid protein comprises one or more amino acid substitutionscompared to a corresponding parental AAV capsid protein, e.g., awild-type capsid protein, and can further comprise from one to about 10amino acid deletions corresponding parental AAV capsid protein, e.g., awild-type capsid protein. In some embodiments, the encoded mutant capsidprotein comprises one or more amino acid substitutions compared to acorresponding parental AAV capsid protein, e.g., a wild-type capsidprotein, and does not comprise any amino acid insertions, e.g., does notcomprise any insertions of amino acids that provide an epitope notpresent in the corresponding AAV capsid protein (e.g., wild-type AAVcapsid protein).

In some embodiments, a subject nucleic acid comprises a nucleotidesequence that comprises from about 1 to about 30 nucleotide differences(e.g., from about 1 to about 5, from about 5 to about 10, from about 10to about 20, or from about 20 to about 30 nucleotide differences) from anucleotide sequence as set forth in any one of SEQ ID NOs:2, 3, 6, 8,10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, and 40, orany one of the nucleotide sequences set forth in FIGS. 4A-G, FIGS. 6A-J,or FIGS. 8A-G. In some embodiments, a subject nucleic acid comprises anucleotide sequence that hybridizes under stringent hybridizationconditions to a nucleic acid having a nucleotide sequence as set forthin any one of SEQ ID NOs:2, 3, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26,28, 30, 32, 34, 36, 38, and 40, or any one of the nucleotide sequencesset forth in FIGS. 4A-G, FIGS. 6A-J, or FIGS. 8A-G. In some embodiments,a subject nucleic acid comprises a nucleotide sequence that is at leastabout 80%, at least about 85%, at least about 90%, at least about 95%,at least about 98%, at least about 99%, or more, identical to anucleotide sequence as set forth in any one of SEQ ID NOs:2, 3, 6, 8,10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, and 40, orany one of the nucleotide sequences set forth in FIGS. 4A-G, FIGS. 6A-J,or FIGS. 8A-G. In some embodiments, a subject nucleic acid comprises anucleic acid having a nucleotide sequence as set forth in any one of SEQID NOs:2, 3, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34,36, 38, and 40, or any one of the nucleotide sequences set forth inFIGS. 4A-G, FIGS. 6A-J, or FIGS. 8A-G.

In some embodiments, a subject nucleic acid comprises a nucleotidesequence that encodes a variant AAV capsid protein that comprises anamino acid sequence that has at least about 85%, at least about 90%, atleast about 95%, at least about 98%, or at least about 99%, or greater,amino acid sequence identity to the amino acid sequence as set forth inSEQ ID NO:42, or as set forth in FIGS. 10A-B (AAV2.5T), where the VP1 ofthe mutant AAV virion comprises an amino acid sequence that has from1-5, from 5-10, from 10-20, from 20 to 25, or from 25-45 amino aciddifferences from the amino acid sequence as set forth in SEQ ID NO:43(AAVS). In some embodiments, a subject nucleic acid comprises anucleotide sequence that encodes a variant AAV capsid protein thatcomprises an A581T substitution compared to the amino acid sequence ofan AAVS capsid protein set forth in SEQ ID NO:43.

In some embodiments, a subject nucleic acid comprises a nucleotidesequence that encodes a variant AAV capsid protein that comprises anamino acid sequence that has at least about 85%, at least about 90%, atleast about 95%, at least about 98%, or at least about 99%, or greater,amino acid sequence identity to the amino acid sequence as set forth inany one of SEQ ID NOs:44-58, where the VP1 of the mutant AAV virioncomprises from 1 to 5, from 5 to 10, or from 10 to 15 amino acidsubstitutions compared to the amino acid sequence of AAV2 as set forthin SEQ ID NO:5. In some embodiments, a subject nucleic acid comprises anucleotide sequence that encodes a variant AAV capsid protein thatcomprises an R459G substitution compared to the amino acid sequence ofAAV2 as set forth in SEQ ID NO:5. In some embodiments, a subject nucleicacid comprises a nucleotide sequence that encodes a variant AAV capsidprotein that comprises a V7081 substitution and a P250S substitutioncompared to the amino acid sequence of AAV2 as set forth in SEQ ID NO:5.

In some embodiments, a subject nucleic acid comprises a nucleotidesequence that encodes a variant AAV capsid protein that comprises anamino acid sequence that has at least about 85%, at least about 90%, atleast about 95%, at least about 98%, or at least about 99%, or greater,amino acid sequence identity to the amino acid sequence as set forth inSEQ ID NO:59, where the VP1 of the mutant AAV virion comprises from 1 to5, from 5 to 10, or from 10 to 15 amino acid substitutions compared tothe amino acid sequence of AAV2 as set forth in SEQ ID NO:5. In someembodiments, a subject nucleic acid comprises a nucleotide sequence thatencodes a variant AAV capsid protein that comprises an S85Gsubstitutionand an R459G substitution compared to the amino acid sequence of AAV2 asset forth in SEQ ID NO:5.

In some embodiments, a subject nucleic acid comprises, in addition to anucleotide sequence that encodes a variant AAV capsid protein (asdescribed above), a gene-targeting cassette that provides for increasedfrequency of homologous recombination of the subject nucleic acid with atarget nucleotide sequence in the genome of a host cell (e.g., a cell inan individual).

The present invention further provides host cells, e.g., isolated(genetically modified) host cells, comprising a subject nucleic acid. Asubject host cell is typically an isolated cell, e.g., a cell in invitro culture. A subject host cell is useful for producing a subjectrAAV virion, as described below. Where a subject host cell is used toproduce a subject rAAV virion, it is referred to as a “packaging cell.”In some embodiments, a subject host cell is stably genetically modifiedwith a subject nucleic acid. In other embodiments, a subject host cellis transiently genetically modified with a subject nucleic acid.

A subject nucleic acid is introduced stably or transiently into a hostcell, using established techniques, including, but not limited to,electroporation, calcium phosphate precipitation, liposome-mediatedtransfection, and the like. For stable transformation, a subject nucleicacid will generally further include a selectable marker, e.g., any ofseveral well-known selectable markers such as neomycin resistance, andthe like.

A subject host cell is generated by introducing a subject nucleic acidinto any of a variety of cells, e.g., mammalian cells, including, e.g.,murine cells, and primate cells (e.g., human cells). Suitable mammaliancells include, but are not limited to, primary cells and cell lines,where suitable cell lines include, but are not limited to, 293 cells,COS cells, HeLa cells, Vero cells, 3T3 mouse fibroblasts, C3H10T1/2fibroblasts, CHO cells, and the like.

In some embodiments, a subject host cell includes, in addition to anucleic acid comprising a nucleotide sequence encoding a mutant capsidprotein, a nucleic acid that comprises a nucleotide sequence encodingone or more AAV rep proteins. In other embodiments, a subject host cellfurther comprises an rAAV vector, as described below. As described inmore detail below, an rAAV virion is generated using a subject hostcell.

rAAV Virions

A mutant capsid protein may be incorporated into an AAV that comprises aheterologous nucleic acid that provides for production of a heterologousgene product (e.g., a heterologous nucleic acid or a heterologousprotein). A subject recombinant AAV virion (“rAAV virion”) comprises amutant capsid protein, and includes a heterologous nucleic acid thatencodes a heterologous gene product. Thus, the present inventionprovides rAAV virions that comprise a mutant capsid protein, asdescribed above; and a heterologous nucleic acid. A subject rAAV virionis useful for introducing a gene product into an individual.

A subject rAAV virion comprises a mutant capsid protein, as describedabove. By virtue of comprising a mutant capsid protein, a subject rAAVvirion exhibits one or more of the following properties: 1) increasedheparin binding affinity relative to wild-type AAV; 2) decreased heparinbinding affinity relative to wild-type AAV; 3) increased infectivity ofa cell that is non-permissive to infection with AAV; 4) increasedevasion of neutralizing antibodies; and 5) increased ability to cross anendothelial cell layer.

In some embodiments, a subject rAAV virion exhibits increased bindingaffinity to heparin relative to a wild-type AAV virion. In theseembodiments, a capsid protein encoded by a subject rAAV virion exhibitsat least about 10%, at least about 15%, at least about 20%, at leastabout 25%, at least about 30%, at least about 35%, at least about 40%,at least about 45%, at least about 50%, at least about 55%, at leastabout 60%, at least about 65%, at least about 70%, at least about 75%,at least about 80%, at least about 85%, at least about 90%, at leastabout 95%, at least about 2-fold, at least 2.5-fold, at least about5-fold, at least about 10-fold, at least about 15-fold, at least about50 fold, at least about 75-fold, or at least about 100-fold or more,higher affinity for heparin than wild-type AAV capsid. Typically,wild-type AAV elutes from a heparin affinity chromatography medium witha NaCl concentration in a range of from about 450 mM to about 550 mM. Insome embodiments, a subject rAAV virion elutes from a heparin affinitychromatography medium with a NaCl concentration of greater than about550 mM, e.g., from about 575 mM NaCl to about 600 mM NaCl, from about600 mM NaCl to about 625 mM NaCl, from about 625 mM NaCl to about 650 mMNaCl, from about 650 mM NaCl to about 675 mM NaCl, from about 675 mMNaCl to about 700 mM NaCl, from about 700 mM NaCl to about 725 mM NaCl,from about 725 mM NaCl to about 750 mM NaCl, from about 750 mM NaCl toabout 775 mM NaCl, or from about 775 mM NaCl to about 800 mM NaCl, orhigher.

In other embodiments, a subject rAAV virion exhibits a lower affinityfor heparin than wild-type AAV. In these embodiments, a subject rAAVvirion has at least about 10%, at least about 15%, at least about 20%,at least about 25%, at least about 30%, at least about 35%, at leastabout 40%, at least about 45%, at least about 50%, at least about 55%,at least about 60%, at least about 65%, at least about 70%, at leastabout 75%, at least about 80%, or at least about 85% lower affinity forheparin than wild-type AAV. In some embodiments, a subject rAAV virionelutes from a heparin affinity chromatography medium with concentrationof NaCl in the range of from about 440 mM NaCl to about 400 mM NaCl,from about 400 mM NaCl to about 375 mM NaCl, from about 375 mM NaCl toabout 350 mM NaCl, from about 350 mM NaCl to about 325 mM NaCl, fromabout 325 mM NaCl to about 300 mM NaCl, from about 300 mM NaCl to about275 mM NaCl, from about 275 mM NaCl to about 250 mM NaCl, from about 250mM NaCl to about 225 mM NaCl, from about 225 mM NaCl to about 200 mMNaCl or lower.

Heparin binding affinity can be determined using any known assay. Forexample, affinity of variant capsids for heparan sulfate can be measuredby binding viral particles to immobilized heparin. See, e.g., Qui et al.(2000) Virology 269:137-147.

In some embodiments, a subject rAAV virion exhibits increased resistanceto neutralizing antibodies compared to wild-type AAV or AAV comprising awild-typo capsid protein. In these embodiments, a subject rAAV virionhas from about 10-fold to about 10,000-fold greater resistance toneutralizing antibodies than wt AAV, e.g., a subject rAAV virion hasfrom about 10-fold to about 25-fold, from about 25-fold to about50-fold, from about 50-fold to about 75-fold, from about 75-fold toabout 100-fold, from about 100-fold to about 150-fold, from about150-fold to about 200-fold, from about 200-fold to about 250-fold, fromabout 250-fold to about 300-fold, at least about 350-fold, at leastabout 400-fold, from about 400-fold to about 450-fold, from about450-fold to about 500-fold, from about 500-fold to about 550-fold, fromabout 550-fold to about 600-fold, from about 600-fold to about 700-fold,from about 700-fold to about 800-fold, from about 800-fold to about900-fold, from about 900-fold to about 1000-fold, from about 1,000-foldto about 2,000-fold, from about 2,000-fold to about 3,000-fold, fromabout 3,000-fold to about 4,000-fold, from about 4,000-fold to about5,000-fold, from about 5,000-fold to about 6,000-fold, from about6,000-fold to about 7,000-fold, from about 7,000-fold to about8,000-fold, from about 8,000-fold to about 9,000-fold, or from about9,000-fold to about 10,000-fold greater resistance to neutralizingantibodies than a wild-type AAV or an AAV comprising a wild-type capsidprotein.

In some embodiments, a subject rAAV virion exhibits decreased binding toa neutralizing antibody that binds a wild-type AAV capsid protein. Forexample, a subject mutant rAAV virion exhibits from about 10-fold toabout 10,000-fold reduced binding to a neutralizing antibody that bindsa wild-type AAV capsid protein. For example, a subject mutant rAAVvirion exhibits from about 10-fold to about 25-fold, from about 25-foldto about 50-fold, from about 50-fold to about 75-fold, from about75-fold to about 100-fold, from about 100-fold to about 150-fold, fromabout 150-fold to about 200-fold, from about 200-fold to about 250-fold,from about 250-fold to about 300-fold, at least about 350-fold, at leastabout 400-fold, from about 400-fold to about 450-fold, from about450-fold to about 500-fold, from about 500-fold to about 550-fold, fromabout 550-fold to about 600-fold, from about 600-fold to about 700-fold,from about 700-fold to about 800-fold, from about 800-fold to about900-fold, from about 900-fold to about 1000-fold, from about 1,000-foldto about 2,000-fold, from about 2,000-fold to about 3,000-fold, fromabout 3,000-fold to about 4,000-fold, from about 4,000-fold to about5,000-fold, from about 5,000-fold to about 6,000-fold, from about6,000-fold to about 7,000-fold, from about 7,000-fold to about8,000-fold, from about 8,000-fold to about 9,000-fold, or from about9,000-fold to about 10,000-fold reduced binding to a neutralizingantibody that binds a wild-type capsid AAV protein, compared to thebinding affinity of the antibody to wild-type AAV capsid protein.

In some embodiments, an anti-AAV neutralizing antibody binds to asubject rAAV virion with an affinity of less than about 10⁻⁷ M, lessthan about 5×10⁻⁶ M, less than about 10⁻⁶ M, less than about 5×10⁻⁵ M,less than about 10⁻⁵ M, less than about 10⁻⁴ M, or lower.

A subject rAAV virion that exhibits reduced binding to neutralizingantibodies has increased residence time in the body, compared to theresidence time of an AAV virion comprising wild-type capsid proteins.Thus, e.g., a subject rAAV virion has at least about 25%, at least about50%, at least about 75%, at least about 2-fold, at least about 2.5-fold,at least about 3-fold, at least about 4-fold, at least about 5-fold atleast about 10-fold, at least about 15-fold, at least about 20-fold, atleast about 50-fold, or more, increased residence time in vivo comparedto the residence time of an AAV virion comprising wild-type capsidproteins.

Whether a given mutant rAAV virion exhibits reduced binding to aneutralizing antibody and/or increased resistance to neutralizingantibody can be determined using any known assay, including the assaydescribed in the Example. For example, mutant rAAV virion is contactedwith a permissive cell type, e.g., 293 cells, in the presence ofneutralizing antibody. A control sample contains the cells, mutant rAAVvirion, and no neutralizing antibody. After a suitable time, the cellsare contacted with adenovirus, and rAAV particles are detected. Thelevel of rAAV particles is compared to the amount of rAAV particles thatare generated in the absence of neutralizing antibody.

In some embodiments, a subject rAAV virion exhibits increased ability toinfect a cell that is relatively refractory to AAV infection (e.g., anon-permissive cell). In these embodiments, a subject mutant AAVexhibits at least about 10%, at least about 20%, at least about 30%, atleast about 40%, at least about 50%, at least about 60%, at least about70%, at least about 80%, at least about 90%, at least about 2-fold, atleast about 4-fold, at least about 10-fold, at least about 20-fold, orat least about 50-fold, or more, greater infectivity of a non-permissivecell than a wild-type AAV or an rAAV virion comprising wild-type capsidprotein.

Examples of cells that are relatively refractory to AAV infectioninclude, but are not limited to stem cells, hepatocytes, and lungepithelial cells.

The term “stem cell” is used herein to refer to a mammalian cell thathas the ability both to self-renew, and to generate differentiatedprogeny (see, e.g., Morrison et al. (1997) Cell 88:287-298). Generally,stem cells also have one or more of the following properties: an abilityto undergo asynchronous, or symmetric replication, that is where the twodaughter cells after division can have different phenotypes; extensiveself-renewal capacity; capacity for existence in a mitotically quiescentform; and clonal regeneration of all the tissue in which they exist, forexample the ability of hematopoietic stem cells to reconstitute allhematopoietic lineages. “Progenitor cells” differ from stem cells inthat they typically do not have the extensive self-renewal capacity, andoften can only regenerate a subset of the lineages in the tissue fromwhich they derive, for example only lymphoid, or erythroid lineages in ahematopoietic setting.

Stem cells may be characterized by both the presence of markersassociated with specific epitopes identified by antibodies and theabsence of certain markers as identified by the lack of binding ofspecific antibodies. Stem cells may also be identified by functionalassays both in vitro and in vivo, particularly assays relating to theability of stem cells to give rise to multiple differentiated progeny.

Stem cells of interest include hematopoietic stem cells and progenitorcells derived therefrom (U.S. Pat. No. 5,061,620); neural crest stemcells (see Morrison et al. (1999) Cell 96:737-749); adult neural stemcells and neural progenitor cells; embryonic stem cells; mesenchymalstem cells; mesodermal stem cells; etc. Other hematopoietic “progenitor”cells of interest include cells dedicated to lymphoid lineages, e.g.immature T cell and B cell populations.

In some embodiments, a subject rAAV virion exhibits increased ability tocross an endothelial cell layer. For example, in these embodiments, asubject rAAV virion exhibits at least about 10%, at least about 20%, atleast about 30%, at least about 40%, at least about 50%, at least about60%, at least about 70%, at least about 80%, at least about 90%, atleast about 2-fold, at least about 5-fold, at least about 10-fold, atleast about 25-fold, or at least about 50-fold increase in ability tocross an endothelial cell layer.

Whether a given rAAV virion exhibits an increased ability to cross anendothelial cell layer can be determined experimentally using well-knownsystems.

A subject rAAV virion comprises a mutation in at least one capsidprotein (e.g., at least one of VP1, VP2, and VP3). Thus, at least one ofVP1, VP2, and VP3 has at least one amino acid substitution compared towild-type AAV capsid protein. In some embodiments, at least one of VP1,VP2, and VP3 has from one to about 25 amino acid substitutions comparedto wild-type AAV VP1, VP2, and VP3, e.g., from about one to about 5,from about 5 to about 10, from about 10 to about 15, from about 15 toabout 20, or from about 20 to about 25 amino acid substitutions comparedto wild-type AAV VP1, VP2, and VP3. Alternatively, a subject rAAV virioncomprises one or more amino acid deletions and/or insertions in at leastone capsid protein relative to wild-type capsid protein. In someembodiments, a subject rAAV virion comprises one or more amino acidsubstitutions and/or deletions and/or insertions in a capsid proteinrelative to a wild-type capsid protein.

In some embodiments, a subject rAAV virion exhibits reduced binding toneutralizing antibody compared to wild-type AAV, and comprises a VP1that has an amino acid sequence that has at least about 85%, at leastabout 90%, at least about 95%, at least about 98%, or at least about99%, or greater, amino acid sequence identity to an amino acid sequenceas set forth in one of SEQ ID NOs:7, 9, 11, 13, 15, 17, 19, 21, 23, 25,27, 29, 31, and 33, or as set forth in FIGS. 5A-C or FIGS. 7A-D. In someembodiments, a subject rAAV virion exhibits reduced binding toneutralizing antibody compared to wild-type AAV, and comprises a VP1that has an amino acid sequence that has from 1-5, from 5-10, or from10-20 amino acid differences from an amino acid sequence as set forth inone of SEQ ID NOs: 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, and33, or as set forth in FIGS. 5A-C or FIGS. 7A-D. In some embodiments, asubject rAAV virion exhibits reduced binding to neutralizing antibodycompared to wild-type AAV, and comprises a VP1 that has an amino acidsequence as set forth in one of SEQ ID NOs: 7, 9, 11, 13, 15, 17, 19,21, 23, 25, 27, 29, 31, and 33, or as set forth in FIGS. 5A-C or FIGS.7A-D.

In some embodiments, a subject rAAV virion exhibits increased heparansulfate affinity compared to wild-type AAV, and comprises a VP1 that hasan amino acid sequence that has at least about 85%, at least about 90%,at least about 95%, at least about 98%, or at least about 99%, orgreater, amino acid sequence identity to an amino acid sequence as setforth in SEQ ID NO:35 or SEQ ID NO:37, or as set forth in FIGS. 9A-C(D14H1 or D14L3). In some embodiments, a subject rAAV virion exhibitsincreased heparan sulfate affinity compared to wild-type AAV, andcomprises a VP1 that has an amino acid sequence that has from 1-5, from5-10, or from 10-20 amino acid differences from an amino acid sequenceas set forth in SEQ ID NO:35 or SEQ ID NO:37, or as set forth in FIGS.9A-C (D14H1 or D14L3). In some embodiments, a subject rAAV virionexhibits increased heparan sulfate affinity compared to wild-type AAV,and comprises a VP1 that has an amino acid sequence as set forth in SEQID NO:35 or SEQ ID NO:37, or as set forth in FIGS. 9A-C (D14H1 orD14L3).

In some embodiments, a subject rAAV virion exhibits reduced heparansulfate affinity compared to wild-type AAV, and comprises a VP1 that hasan amino acid sequence that has at least about 85%, at least about 90%,at least about 95%, at least about 98%, or at least about 99%, orgreater, amino acid sequence identity to an amino acid sequence as setforth in SEQ ID NO:39 or SEQ ID NO:41, or as set forth in FIGS. 9A-C(P1BH1 or P1BH2). In some embodiments, a subject rAAV virion exhibitsreduced heparan sulfate affinity compared to wild-type AAV, andcomprises a VP1 that has an amino acid sequence that has from 1-5, from5-10, or from 10-20 amino acid differences from an amino acid sequenceas set forth in SEQ ID NO:39 or SEQ ID NO:41, or as set forth in FIGS.9A-C (P1BH1 or P1BH2). In some embodiments, a subject rAAV virionexhibits decreased heparan sulfate affinity compared to wild-type AAV,and comprises a VP1 that has an amino acid sequence as set forth in SEQID NO:39 or SEQ ID NO:41, or as set forth in FIGS. 9A-C (P1BH1 orP1BH2).

In some embodiments, a subject rAAV virion exhibits increasedinfectivity of a non-permissive cell (e.g., a lung epithelial cell)compared to the infectivity of the non-permissive cell by acorresponding parental AAV virion (e.g., wild-type AAV), and comprises avariant AAV capsid protein that comprises an amino acid sequence thathas at least about 85%, at least about 90%, at least about 95%, at leastabout 98%, or at least about 99%, or greater, amino acid sequenceidentity to the amino acid sequence as set forth in SEQ ID NO:42, or asset forth in FIGS. 10A-B (AAV2.5T), where the VP1 of the rAAV virioncomprises an amino acid sequence that has from 1-5, from 5-10, from10-20, from 20 to 25, or from 25-45 amino acid differences from theamino acid sequence as set forth in SEQ ID NO:43 (AAVS). In someembodiments, a subject rAAV virion exhibits increased infectivity of anon-permissive cell (e.g., a lung epithelial cell) compared to theinfectivity of the non-permissive cell by a corresponding parental AAVvirion (e.g., wild-type AAV), and comprises a variant AAV capsid proteinthat comprises an A581T substitution compared to the amino acid sequenceof an AAVS capsid protein set forth in SEQ ID NO:43.

In some embodiments, a subject rAAV virion exhibits increasedinfectivity of a non-permissive cell (e.g., a stem cell) compared to theinfectivity of the non-permissive cell by a corresponding parental AAVvirion (e.g., wild-type AAV), and comprises a variant AAV capsid proteinthat comprises an amino acid sequence that has at least about 85%, atleast about 90%, at least about 95%, at least about 98%, or at leastabout 99%, or greater, amino acid sequence identity to the amino acidsequence as set forth in any one of SEQ ID NOs:44-58, where the VP1 ofthe rAAV virion comprises from 1 to 5, from 5 to 10, or from 10 to 15amino acid substitutions compared to the amino acid sequence of AAV2 asset forth in SEQ ID NO:5. In some embodiments, a subject rAAV virionexhibits increased infectivity of a non-permissive cell (e.g., a stemcell) compared to the infectivity of the non-permissive cell by acorresponding parental AAV virion (e.g., wild-type AAV), and comprises avariant AAV capsid protein that comprises an R459G substitution comparedto the amino acid sequence of AAV2 as set forth in SEQ ID NO:5. In someembodiments, a subject rAAV virion exhibits increased infectivity of anon-permissive cell (e.g., a stem cell) compared to the infectivity ofthe non-permissive cell by a corresponding parental AAV virion (e.g.,wild-type AAV), and comprises a variant AAV capsid protein thatcomprises a V7081 substitution and a P250S substitution compared to theamino acid sequence of AAV2 as set forth in SEQ ID NO:5.

In some embodiments, a subject mutant AAV virion exhibits increasedinfectivity of a non-permissive stem cell compared to a correspondingparental AAV (e.g., a wild-type AAV), and comprises a VP1 that comprisesan amino acid sequence that has at least about 85%, at least about 90%,at least about 95%, at least about 98%, or at least about 99%, orgreater, amino acid sequence identity to the amino acid sequence as setforth in SEQ ID NO: 59, where the VP1 of the mutant AAV virion comprisesfrom 1 to 5, from 5 to 10, or from 10 to 15 amino acid substitutionscompared to the amino acid sequence of AAV2 as set forth in SEQ ID NO:5.In some embodiments, a subject mutant AAV virion exhibits increasedinfectivity of a non-permissive stem cell compared to wild-type AAV2,and comprises an S85G substitution and an R459G substitution compared tothe amino acid sequence of AAV2 as set forth in SEQ ID NO:5.

Generation of Subject rAAV Virions

By way of introduction, it is typical to employ a host or “producer”cell for rAAV vector replication and packaging. Such a producer cell(usually a mammalian host cell) generally comprises or is modified tocomprise several different types of components for rAAV production. Thefirst component is a recombinant adeno-associated viral (rAAV) vectorgenome (or “rAAV pro-vector”) that can be replicated and packaged intovector particles by the host packaging cell. The rAAV pro-vector willnormally comprise a heterologous polynucleotide (or “transgene”), withwhich it is desired to genetically alter another cell in the context ofgene therapy (since the packaging of such a transgene into rAAV vectorparticles can be effectively used to deliver the transgene to a varietyof mammalian cells). The transgene is generally flanked by two AAVinverted terminal repeats (ITRs) which comprise sequences that arerecognized during excision, replication and packaging of the AAV vector,as well as during integration of the vector into a host cell genome.

A second component is a helper virus that can provide helper functionsfor AAV replication. Although adenovirus is commonly employed, otherhelper viruses can also be used as is known in the art. Alternatively,the requisite helper virus functions can be isolated genetically from ahelper virus and the encoding genes can be used to provide helper virusfunctions in trans. The AAV vector elements and the helper virus (orhelper virus functions) can be introduced into the host cell eithersimultaneously or sequentially in any order.

The final components for AAV production to be provided in the producercell are “AAV packaging genes” such as AAV rep and cap genes thatprovide replication and encapsidation proteins, respectively. Severaldifferent versions of AAV packaging genes can be provided (includingrep-cap cassettes and separate rep and/or cap cassettes in which the repand/or cap genes can be left under the control of the native promotersor operably linked to heterologous promoters. Such AAV packaging genescan be introduced either transiently or stably into the host packagingcell, as is known in the art and described in more detail below.

1. rAAV Vector

A subject rAAV virion, including the heterologous DNA of interest (where“heterologous DNA of interest” is also referred to herein as“heterologous nucleic acid”), can be produced using standardmethodology, known to those of skill in the art. The methods generallyinvolve the steps of (1) introducing a subject rAAV vector into a hostcell; (2) introducing an AAV helper construct into the host cell, wherethe helper construct includes AAV coding regions capable of beingexpressed in the host cell to complement AAV helper functions missingfrom the AAV vector; (3) introducing one or more helper viruses and/oraccessory function vectors into the host cell, wherein the helper virusand/or accessory function vectors provide accessory functions capable ofsupporting efficient recombinant AAV (“rAAV”) virion production in thehost cell; and (4) culturing the host cell to produce rAAV virions. TheAAV expression vector, AAV helper construct and the helper virus oraccessory function vector(s) can be introduced into the host cell,either simultaneously or serially, using standard transfectiontechniques.

AAV expression vectors are constructed using known techniques to atleast provide as operatively linked components in the direction oftranscription, control elements including a transcriptional initiationregion, the DNA of interest and a transcriptional termination region.The control elements are selected to be functional in a mammalian musclecell. The resulting construct which contains the operatively linkedcomponents is bounded (5′ and 3′) with functional AAV ITR sequences.

The nucleotide sequences of AAV ITR regions are known. See, e.g., Kotin,R. M. (1994) Human Gene Therapy 5:793-801; Berns, K. I. “Parvoviridaeand their Replication” in Fundamental Virology, 2nd Edition, (B. N.Fields and D. M. Knipe, eds.) for the AAV-2 sequence. AAV ITRs used inthe vectors of the invention need not have a wild-type nucleotidesequence, and may be altered, e.g., by the insertion, deletion orsubstitution of nucleotides. Additionally, AAV ITRs may be derived fromany of several AAV serotypes, including without limitation, AAV-1,AAV-2, AAV-3, AAV-4, AAV-5, AAV-7, etc. Furthermore, 5′ and 3′ ITRswhich flank a selected nucleotide sequence in an AAV expression vectorneed not necessarily be identical or derived from the same AAV serotypeor isolate, so long as they function as intended, i.e., to allow forexcision and rescue of the sequence of interest from a host cell genomeor vector, and to allow integration of the DNA molecule into therecipient cell genome when AAV Rep gene products are present in thecell. ITRs allow replication of the vector sequence in the presence ofan appropriate mixture of Rep proteins. ITRs also allow for theincorporation of the vector sequence into the capsid to generate an AAVparticle.

A suitable heterologous DNA molecule (also referred to herein as a“heterologous nucleic acid”) for use in a subject rAAV vector willgenerally be less than about 5 kilobases (kb) in size and will include,for example, a gene (a nucleotide sequence) that encodes a protein thatis defective or missing from a recipient subject; a gene that encodes aprotein having a desired biological or therapeutic effect (e.g., anantibacterial, antiviral or antitumor function); a nucleotide sequencethat encodes an RNA that inhibits or reduces production of a deleteriousor otherwise undesired protein; a nucleotide sequence that encodes anantigenic protein; or a nucleotide sequence that encodes an RNA thatinhibits or reduces production of a protein.

Suitable heterologous nucleic acids include, but are not limited to,those encoding proteins used for the treatment of endocrine, metabolic,hematologic, cardiovascular, neurologic, musculoskeletal, urologic,pulmonary and immune disorders, including such disorders as inflammatorydiseases, autoimmune, chronic and infectious diseases, such as acquiredimmunodeficiency syndrome (AIDS), cancer, hypercholestemia, insulindisorders such as diabetes, growth disorders, various blood disordersincluding various anemias, thalassemias and hemophilia; genetic defectssuch as cystic fibrosis, Gaucher's Disease, Hurler's Disease, adenosinedeaminase (ADA) deficiency, emphysema, or the like.

Suitable heterologous nucleic acids include, but are not limited to,those encoding any of a variety of proteins, including, but not limitedto: an interferon (e.g., IFN-γ, IFN-α, IFN-ω; IFN-τ); an insulin (e.g.,Novolin, Humulin, Humalog, Lantus, Ultralente, etc.); an erythropoietin(“EPO”; e.g., Procrit®, Eprex®, or Epogen® (epoetin-α); Aranesp®(darbepoietin-α); NeoRecormon®, Epogin® (epoetin-β); and the like); anantibody (e.g., a monoclonal antibody) (e.g., Rituxan® (rituximab);Remicade® (infliximab); Herceptin® (trastuzumab); Humira™ (adalimumab);Xolair® (omalizumab); Bexxar® (tositumomab); Raptiva™ (efalizumab);Erbitux™ (cetuximab); and the like), including an antigen-bindingfragment of a monoclonal antibody; a blood factor (e.g., Activase®(alteplase) tissue plasminogen activator; NovoSeven® (recombinant humanfactor VIIa); Factor VIIa; Factor VIII (e.g., Kogenate®); Factor IX;β-globin; hemoglobin; and the like); a colony stimulating factor (e.g.,Neupogen® (filgrastim; G-CSF); Neulasta (pegfilgrastim); granulocytecolony stimulating factor (G-CSF), granulocyte-monocyte colonystimulating factor, macrophage colony stimulating factor, megakaryocytecolony stimulating factor; and the like); a growth hormone (e.g., asomatotropin, e.g., Genotropin®, Nutropin®, Norditropin®, Saizen®,Serostim®, Humatrope®, etc.; a human growth hormone; and the like); aninterleukin (e.g., IL-1; IL-2, including, e.g., Proleukin®; IL-3, IL-4,IL-5, IL-6, IL-7, IL-8, IL-9; etc.); a growth factor (e.g., Regranex®(beclapermin; PDGF); Fiblast® (trafermin; bFGF); Stemgen® (ancestim;stem cell factor); keratinocyte growth factor; an acidic fibroblastgrowth factor, a stem cell factor, a basic fibroblast growth factor, ahepatocyte growth factor; and the like); a soluble receptor (e.g., aTNF-α-binding soluble receptor such as Enbrel® (etanercept); a solubleVEGF receptor; a soluble interleukin receptor; a soluble γ/δ T cellreceptor; and the like); an enzyme (e.g., α-glucosidase; Cerazyme®(imiglucarase; β-glucocerebrosidase, Ceredase® (alglucerase;); an enzymeactivator (e.g., tissue plasminogen activator); a chemokine (e.g.,IP-10; Mig; Groa/IL-8, RANTES; MIP-1α; MIP-1β; MCP-1; PF-4; and thelike); an angiogenic agent (e.g., vascular endothelial growth factor(VEGF) ; an anti-angiogenic agent (e.g., a soluble VEGF receptor); aprotein vaccine; a neuroactive peptide such as bradykinin,cholecystokinin, gastin, secretin, oxytocin, gonadotropin-releasinghormone, beta-endorphin, enkephalin, substance P, somatostatin,prolactin, galanin, growth hormone-releasing hormone, bombesin,dynorphin, neurotensin, motilin, thyrotropin, neuropeptide Y,luteinizing hormone, calcitonin, insulin, glucagon, vasopressin,angiotensin II, thyrotropin-releasing hormone, vasoactive intestinalpeptide, a sleep peptide, etc.; other proteins such as a thrombolyticagent, an atrial natriuretic peptide, bone morphogenic protein,thrombopoietin, relaxin, glial fibrillary acidic protein, folliclestimulating hormone, a human alpha-1 antitrypsin, a leukemia inhibitoryfactor, a transforming growth factor, an insulin-like growth factor, aluteinizing hormone, a macrophage activating factor, tumor necrosisfactor, a neutrophil chemotactic factor, a nerve growth factor a tissueinhibitor of metalloproteinases; a vasoactive intestinal peptide,angiogenin, angiotropin, fibrin; hirudin; a leukemia inhibitory factor;an IL-1 receptor antagonist (e.g., Kineret® (anakinra)); an ion channel,e.g., cystic fibrosis transmembrane conductance regulator (CFTR);dystrophin; utrophin, a tumor suppressor; lysosomal enzyme acida-glucosidase (GAA); and the like. Suitable nucleic acids also includethose that encode a functional fragment of any of the aforementionedproteins; and nucleic acids that encode functional variants of any ofthe aforementioned proteins.

Suitable heterologous nucleic acids also include those that encodeantigenic proteins. A subject rAAV that comprises a heterologous nucleicacid that encodes an antigenic protein is suitable for stimulating animmune response to the antigenic protein in a mammalian host. Theantigenic protein is derived from an autoantigen, an allergen, atumor-associated antigen, a pathogenic virus, a pathogenic bacterium, apathogenic protozoan, a pathogenic helminth, or any other pathogenicorganism that infects a mammalian host. As used herein, the term “anucleic acid encoding an antigenic protein derived from” includesnucleic acids encoding wild-type antigenic proteins, e.g., a nucleicacid isolated from a pathogenic virus that encodes a viral protein;synthetic nucleic acids generated in the laboratory that encodeantigenic proteins that are identical in amino acid sequence to anaturally-occurring antigenic protein; synthetic nucleic acids generatedin the laboratory that encode antigenic proteins that differ in aminoacid sequence (e.g., by from one amino acid to about 15 amino acids)from a naturally-occurring antigenic protein, but that nonethelessinduce an immune response to the corresponding naturally-occurringantigenic protein; synthetic nucleic acids generated in the laboratorythat encode fragments of antigenic proteins (e.g., fragments of fromabout 5 amino acids to about 50 amino acids, which fragments comprisesone or more antigenic epitopes), which fragments induce an immuneresponse to the corresponding naturally-occurring antigenic protein;etc.

Similarly, an antigenic protein “derived from” an autoantigen, anallergen, a tumor-associated antigen, a pathogenic virus, a pathogenicbacterium, a pathogenic protozoan, a pathogenic helminth, or any otherpathogenic organism that infects a mammalian host, includes proteinsthat are identical in amino acid sequence to a naturally-occurringantigenic protein, and proteins that differ in amino acid sequence(e.g., by from one amino acid to about 15 amino acids) from anaturally-occurring antigenic protein, but that nonetheless induce animmune response to the corresponding naturally-occurring antigenicprotein; and fragments of antigenic proteins (e.g., fragments of fromabout 5 amino acids to about 50 amino acids, which fragments comprisesone or more antigenic epitopes), which fragments induce an immuneresponse to the corresponding naturally-occurring antigenic protein.

In some embodiments, an immune response to an antigenic protein encodedby a subject rAAV will stimulate a protective immune response to apathogenic organism that displays the antigenic protein or antigenicepitope (or a protein or an epitope that is cross-reactive with therAAV-encoded antigenic protein or antigenic epitopes) in the mammalianhost. In some embodiments, a cytotoxic T lymphocyte (CTL) response tothe rAAV-encoded antigenic protein will be induced in the mammalianhost. In other embodiments, a humoral response to the rAAV-encodedantigenic protein will be induced in the mammalian host, such thatantibodies specific to the antigenic protein are generated. In manyembodiments, a TH1 immune response to the rAAV-encoded antigenic proteinwill be induced in the mammalian host. Suitable antigenic proteinsinclude tumor-associated antigens, viral antigens, bacterial antigens,and protozoal antigens; and antigenic fragments thereof. In someembodiments, the antigenic protein is derived from an intracellularpathogen. In other embodiments, the antigenic protein is a self-antigen.In yet other embodiments, the antigenic protein is an allergen.

Tumor-specific antigens include, but are not limited to, any of thevarious MAGEs (Melanoma-Associated Antigen E), including MAGE 1 (e.g.,GenBank Accession No. M77481), MAGE 2 (e.g., GenBank Accession No.U03735), MAGE 3, MAGE 4, etc.; any of the various tyrosinases; mutantras; mutant p53 (e.g., GenBank Accession No. X54156 and AA494311); andp97 melanoma antigen (e.g., GenBank Accession No. M12154). Othertumor-specific antigens include the Ras peptide and p53 peptideassociated with advanced cancers, the HPV 16/18 and E6/E7 antigensassociated with cervical cancers, MUCI1-KLH antigen associated withbreast carcinoma (e.g., GenBank Accession No. J03651), CEA(carcinoembryonic antigen) associated with colorectal cancer (e.g.,GenBank Accession No. X98311), gp100 (e.g., GenBank Accession No.573003) or MART1 antigens associated with melanoma, and the PSA antigenassociated with prostate cancer (e.g., GenBank Accession No. X14810).The p53 gene sequence is known (See e.g., Harris et al. (1986) Mol.Cell. Biol., 6:4650-4656) and is deposited with GenBank under AccessionNo. M14694. Thus, the present invention can be used asimmunotherapeutics for cancers including, but not limited to, cervical,breast, colorectal, prostate, lung cancers, and for melanomas.

Viral antigens are derived from known causative agents responsible fordiseases including, but not limited to, measles, mumps, rubella,poliomyelitis, hepatitis A, B (e.g., GenBank Accession No. E02707), andC (e.g., GenBank Accession No. E06890), as well as other hepatitisviruses, influenza, adenovirus (e.g., types 4 and 7), rabies (e.g.,GenBank Accession No. M34678), yellow fever, Japanese encephalitis(e.g., GenBank Accession No. E07883), dengue (e.g., GenBank AccessionNo. M24444), hantavirus, and human immunodeficiency virus (e.g., GenBankAccession No. U18552).

Suitable bacterial and parasitic antigens include those derived fromknown causative agents responsible for diseases including, but notlimited to, diphtheria, pertussis (e.g., GenBank Accession No. M35274),tetanus (e.g., GenBank Accession No. M64353), tuberculosis, bacterialand fungal pneumonias (e.g., Haemophilus influenzae, Pneumocystiscarinii, etc.), cholera, typhoid, plague, shigellosis, salmonellosis(e.g., GenBank Accession No. L03833), Legionnaire's Disease, Lymedisease (e.g., GenBank Accession No. U59487), malaria (e.g., GenBankAccession No. X53832), hookworm, onchocerciasis (e.g., GenBank AccessionNo. M27807), schistosomiasis (e.g., GenBank Accession No. L08198),trypanosomiasis, leshmaniasis, giardiasis (e.g., GenBank Accession No.M33641), amoebiasis, filariasis (e.g., GenBank Accession No. J03266),borreliosis, and trichinosis.

In some embodiments, e.g., in the context of stem cells, suitableheterologous nucleic acids include, but are not limited to, thoseencoding any of a variety of proteins, including, but not limited to: agrowth factor, a morphogen, a cytokine, a receptor, a protein involvedin intracellular signal transduction, a protein that provides adetectable signal, and a transcription factor.

In some embodiments, e.g., in the context of stem cells, a suitableheterologous nucleic acid includes a heterologous nucleic acidcomprising a nucleotide sequence encoding a protein including, but notlimited to, a growth factor (e.g., epidermal growth factor, fibroblastgrowth factor, vascular endothelial growth factor, neurotrophin, a TGFβfamily member, a Delta family member, a Jagged family member); amorphogen (e.g., a Wnt or Hedgehog protein); a cytokine (e.g., aninterleukin, a tumor necrosis family member, etc.); a receptor (e.g., agrowth factor receptor, a cytokine receptor, a neurotransmitterreceptor, a neutrotrophin receptor, an ion channel, a Notch familymember, a Patched family member, a TGFβ receptor family member, asteroid hormone receptor, etc.); a protein involved in intracellularsignal transduction (e.g., MAPK, MEKK, PI3-kinase, Akt, PKC, PKA, PKG, amember of the Jak family, a member of the Src family, etc.); atranscription factor (e.g., a member of the GATA, Gli, Sp, Hes, Hey,NF-KB, LIM, Olig, Mash, Math, TCF, Elk, Forkhead, histoneacetyltransferase, histone methyltransferase, or histone deacetylasefamilies); and the like.

In some embodiments, e.g., in the context of lung epithelial cells,suitable heterologous nucleic acids include, but are not limited to,those encoding any of a variety of proteins, including, but not limitedto: a cystic fibrosis transmembrane conductance regulator, an immunogen(e.g., an antigen, as described elsewhere herein), and an antiviralprotein. Non-limiting examples of suitable immunogens include a proteinfrom influenza, a Bacillus anthracis polypeptide, a cancer-associatedantigen, and other antigens as described elsewhere herein. In thecontext of lung epithelial cells, a suitable heterologous nucleic acidcomprises a nucleotide sequence encoding an antiviral protein (e.g.,interferon-beta, interferon-gamma, interleukin-2, etc.).

Suitable heterologous nucleic acids that encode heterologous geneproducts include non-translated RNAs, such as an antisense RNA, aribozyme, an RNAi, an shRNA, and an siRNA. Interfering RNA (RNAi)fragments, particularly double-stranded (ds) RNAi, can be used toinhibit gene expression. One approach well known in the art forinhibiting gene expression is short interfering RNA (siRNA) mediatedgene silencing, where the level of expression product of a target geneis reduced by specific double stranded siRNA nucleotide sequences thatare complementary to at least a 19-25 nucleotide long segment (e.g., a20-21 nucleotide sequence) of the target gene transcript, including the5′ untranslated (UT) region, the ORF, or the 3′ UT region. In someembodiments, short interfering RNAs are about 19-25 nt in length. See,e.g., PCT applications WO0/44895, WO99/32619, WO01/75164, WO01/92513,WO01/29058, WO01/89304, WO02/16620, and WO02/29858; and U.S. PatentPublication No. 20040023390 for descriptions of siRNA technology. ThesiRNA can be encoded by a nucleic acid sequence, and the nucleic acidsequence can also include a promoter. The nucleic acid sequence can alsoinclude a polyadenylation signal. In some embodiments, thepolyadenylation signal is a synthetic minimal polyadelylation signal.

Target genes include any gene encoding a target gene product (RNA orprotein) that is deleterious (e.g., pathological); a target gene productthat is malfunctioning; a target gene product. Target gene productsinclude, but are not limited to, huntingtin; hepatitis C virus; humanimmunodeficiency virus; amyloid precursor protein; tau; a protein thatincludes a polyglutamine repeat; a herpes virus (e.g., varicellazoster); any pathological virus; and the like.

As such a subject rAAV that includes a heterologous nucleic acidencoding an siRNA is useful for treating a variety of disorders andconditions, including, but not limited to, neurodegenerative diseases,e.g., a trinucleotide-repeat disease, such as a disease associated withpolyglutamine repeats, e.g., Huntington's disease , spinocerebellarataxia, spinal and bulbar muscular atrophy (SBMA),dentatorubropallidoluysian atrophy (DRPLA), etc.; an acquired pathology(e.g., a disease or syndrome manifested by an abnormal physiological,biochemical, cellular, structural, or molecular biological state) suchas a viral infection, e.g., hepatitis that occurs or may occur as aresult of an HCV infection, acquired immunodeficiency syndrome, whichoccurs as a result of an HIV infection; and the like.

In many embodiments, a heterologous nucleic acid encoding an siRNA isoperably linked to a promoter. Suitable promoters are known thoseskilled in the art and include the promoter of any protein-encodinggene, e.g., an endogenously regulated gene or a constitutively expressedgene. For example, the promoters of genes regulated by cellularphysiological events, e.g., heat shock, oxygen levels and/or carbonmonoxide levels, e.g., in hypoxia, may be operably linked to ansiRNA-encoding nucleic acid.

The selected heterologous nucleotide sequence, such as EPO-encoding ornucleic acid of interest, is operably linked to control elements thatdirect the transcription or expression thereof in the nucleotidesequence in vivo. Such control elements can comprise control sequencesnormally associated with the selected gene (e.g., endogenous cellularcontrol elements). Alternatively, heterologous control sequences can beemployed. Useful heterologous control sequences generally include thosederived from sequences encoding mammalian or viral genes. Examplesinclude, but are not limited to, the SV40 early promoter, mouse mammarytumor virus long terminal repeat (LTR) promoter; adenovirus major latepromoter (Ad MLP); a herpes simplex virus (HSV) promoter, an endogenouscellular promoter that is heterologous to the gene of interest, acytomegalovirus (CMV) promoter such as the CMV immediate early promoterregion (CMVIE), a rous sarcoma virus (RSV) promoter, syntheticpromoters, hybrid promoters, and the like. In addition, sequencesderived from nonviral genes, such as the murine metallothionein gene,will also find use herein. Such promoter sequences are commerciallyavailable from, e.g., Stratagene (San Diego, Calif.).

In some embodiments, cell type-specific or tissue-specific promoter willbe operably linked to the heterologous nucleic acid encoding theheterologous gene product, such that the gene product is producedselectively or preferentially in a particular cell type(s) or tissue(s).In some embodiments, an inducible promoter will be operably linked tothe heterologous nucleic acid.

For example, muscle-specific and inducible promoters, enhancers and thelike, are useful for delivery of a gene product to a muscle cell. Suchcontrol elements include, but are not limited to, those derived from theactin and myosin gene families, such as from the myoD gene family; themyocyte-specific enhancer binding factor MEF-2; control elements derivedfrom the human skeletal actin gene and the cardiac actin gene; musclecreatine kinase sequence elements and the murine creatine kinaseenhancer (mCK) element; control elements derived from the skeletalfast-twitch troponin C gene, the slow-twitch cardiac troponin C gene andthe slow-twitch troponin I gene; hypoxia-inducible nuclear factors;steroid-inducible elements and promoters, such as the glucocorticoidresponse element (GRE); the fusion consensus element for RU486induction; and elements that provide for tetracycline regulated geneexpression.

The AAV expression vector which harbors the DNA molecule of interest(the heterologous DNA) bounded by AAV ITRs, can be constructed bydirectly inserting the selected sequence(s) into an AAV genome which hashad the major AAV open reading frames (“ORFs”) excised therefrom. Otherportions of the AAV genome can also be deleted, so long as a sufficientportion of the ITRs remain to allow for replication and packagingfunctions. Such constructs can be designed using techniques well knownin the art. See, e.g., U.S. Pat. Nos. 5,173,414 and 5,139,941;International Publication Nos. WO 92/01070 (published Jan. 23, 1992) andWO 93/03769 (published Mar. 4, 1993); Lebkowski et al. (1988) Molec.Cell. Biol. 8:3988-3996; Vincent et al. (1990) Vaccines 90 (Cold SpringHarbor Laboratory Press); Carter, B. J. (1992) Current Opinion inBiotechnology 3:533-539; Muzyczka, N. (1992) Current Topics inMicrobiol. and Immunol. 158:97-129; Kotin, R. M. (1994) Human GeneTherapy 5:793-801; Shelling and Smith (1994) Gene Therapy 1:165-169; andZhou et al. (1994) J. Exp. Med. 179:1867-1875.

Alternatively, AAV ITRs can be excised from the viral genome or from anAAV vector containing the same and fused 5′ and 3′ of a selected nucleicacid construct that is present in another vector using standard ligationtechniques, such as those described in Sambrook et al., supra. Forexample, ligations can be accomplished in 20 mM Tris-Cl pH 7.5, 10 mMMgCl₂, 10 mM DTT, 33 μg/ml BSA, 10 mM-50 mM NaCl, and either 40 μM ATP,0.01-0.02 (Weiss) units T4 DNA ligase at 0° C. to 16° C. (for “stickyend” ligation) or 1 mM ATP, 0.3-0.6 (Weiss) units T4 DNA ligase at 14°C. (for “blunt end” ligation). Intermolecular “sticky end” ligations areusually performed at 30-100 μg/ml total DNA concentrations (5-100 nMtotal end concentration). AAV vectors which contain ITRs have beendescribed in, e.g., U.S. Pat. No. 5,139,941. In particular, several AAVvectors are described therein which are available from the American TypeCulture Collection (“ATCC”) under Accession Numbers 53222, 53223, 53224,53225 and 53226.

Additionally, chimeric genes can be produced synthetically to includeAAV ITR sequences arranged 5′ and 3′ of one or more selected nucleicacid sequences. Preferred codons for expression of the chimeric genesequence in mammalian muscle cells can be used. The complete chimericsequence is assembled from overlapping oligonucleotides prepared bystandard methods. See, e.g., Edge, Nature (1981) 292:756; Nambair et al.Science (1984) 223:1299; Jay et al. J. Biol. Chem. (1984) 259:6311.

In order to produce rAAV virions, an AAV expression vector is introducedinto a suitable host cell using known techniques, such as bytransfection. A number of transfection techniques are generally known inthe art. See, e.g., Graham et al. (1973) Virology, 52:456, Sambrook etal. (1989) Molecular Cloning, A Laboratory Manual, Cold Spring HarborLaboratories, New York, Davis et al. (1986) Basic Methods in MolecularBiology, Elsevier, and Chu et al. (1981) Gene 13:197. Particularlysuitable transfection methods include calcium phosphate co-precipitation(Graham et al. (1973) Virol. 52:456-467), direct micro-injection intocultured cells (Capecchi, M. R. (1980) Cell 22:479-488), electroporation(Shigekawa et al. (1988) BioTechnigues 6:742-751), liposome mediatedgene transfer (Mannino et al. (1988) BioTechniques 6:682-690),lipid-mediated transduction (Feigner et al. (1987) Proc. Natl. Acad.Sci. USA 84:7413-7417), and nucleic acid delivery using high-velocitymicroprojectiles (Klein et al. (1987) Nature 327:70-73).

For the purposes of the invention, suitable host cells for producingrAAV virions include microorganisms, yeast cells, insect cells, andmammalian cells, that can be, or have been, used as recipients of aheterologous DNA molecule. The term includes the progeny of the originalcell which has been transfected. Thus, a “host cell” as used hereingenerally refers to a cell which has been transfected with an exogenousDNA sequence. Cells from the stable human cell line, 293 (readilyavailable through, e.g., the American Type Culture Collection underAccession Number ATCC CRL1573) are used in many embodiments.Particularly, the human cell line 293 is a human embryonic kidney cellline that has been transformed with adenovirus type-5 DNA fragments(Graham et al. (1977) J. Gen. Virol. 36:59), and expresses theadenoviral Ela and E1b genes (Aiello et al. (1979) Virology 94:460). The293 cell line is readily transfected, and provides a particularlyconvenient platform in which to produce rAAV virions.

2. AAV Helper Functions

Host cells containing the above-described AAV expression vectors must berendered capable of providing AAV helper functions in order to replicateand encapsidate the nucleotide sequences flanked by the AAV ITRs toproduce rAAV virions. AAV helper functions are generally AAV-derivedcoding sequences which can be expressed to provide AAV gene productsthat, in turn, function in trans for productive AAV replication. AAVhelper functions are used herein to complement necessary AAV functionsthat are missing from the AAV expression vectors. Thus, AAV helperfunctions include one, or both of the major AAV ORFs, namely the rep andcap coding regions, or functional homologues thereof. In the context ofthe instant invention, the cap functions include one or more mutantcapsid proteins, wherein at least one capsid protein comprises at leastone mutation, as described above.

By “AAV rep coding region” is meant the art-recognized region of the AAVgenome which encodes the replication proteins Rep 78, Rep 68, Rep 52 andRep 40. These Rep expression products have been shown to possess manyfunctions, including recognition, binding and nicking of the AAV originof DNA replication, DNA helicase activity and modulation oftranscription from AAV (or other heterologous) promoters. The Repexpression products are collectively required for replicating the AAVgenome. For a description of the AAV rep coding region, see, e.g.,Muzyczka, N. (1992) Current Topics in Microbiol. and Immunol.158:97-129; and Kotin, R. M. (1994) Human Gene Therapy 5:793-801.Suitable homologues of the AAV rep coding region include the humanherpesvirus 6 (HHV-6) rep gene which is also known to mediate AAV-2 DNAreplication (Thomson et al. (1994) Virology 204:304-311).

AAV cap proteins include VP1, VP2, and VP3, wherein at least one of VP1,VP2, and VP3 comprises at least one mutation, as described above.

AAV helper functions are introduced into the host cell by transfectingthe host cell with an AAV helper construct either prior to, orconcurrently with, the transfection of the AAV expression vector. AAVhelper constructs are thus used to provide at least transient expressionof AAV rep and/or cap genes to complement missing AAV functions that arenecessary for productive AAV infection. AAV helper constructs lack AAVITRs and can neither replicate nor package themselves. These constructscan be in the form of a plasmid, phage, transposon, cosmid, virus, orvirion. A number of AAV helper constructs have been described, such asthe commonly used plasmids pAAV/Ad and pIM29+45 which encode both Repand Cap expression products. See, e.g., Samulski et al. (1989) J. Virol.63:3822-3828; and McCarty et al. (1991) J. Virol. 65:2936-2945. A numberof other vectors have been described which encode Rep and/or Capexpression products. See, e.g., U.S. Pat. No. 5,139,941.

Both AAV expression vectors and AAV helper constructs can be constructedto contain one or more optional selectable markers. Suitable markersinclude genes which confer antibiotic resistance or sensitivity to,impart color to, or change the antigenic characteristics of those cellswhich have been transfected with a nucleic acid construct containing theselectable marker when the cells are grown in an appropriate selectivemedium. Several selectable marker genes that are useful in the practiceof the invention include the hygromycin B resistance gene (encodingAminoglycoside phosphotranferase (APH)) that allows selection inmammalian cells by conferring resistance to hygromycin; the neomycinphosphotranferase gene (encoding neomycin phosphotransferase) thatallows selection in mammalian cells by conferring resistance to G418;and the like. Other suitable markers are known to those of skill in theart.

3. AAV Accessory Functions

The host cell (or packaging cell) must also be rendered capable ofproviding non AAV derived functions, or “accessory functions,” in orderto produce rAAV virions. Accessory functions are non AAV derived viraland/or cellular functions upon which AAV is dependent for itsreplication. Thus, accessory functions include at least those non AAVproteins and RNAs that are required in AAV replication, including thoseinvolved in activation of AAV gene transcription, stage specific AAVmRNA splicing, AAV DNA replication, synthesis of Cap expression productsand AAV capsid assembly. Viral-based accessory functions can be derivedfrom any of the known helper viruses.

Particularly, accessory functions can be introduced into and thenexpressed in host cells using methods known to those of skill in theart. Commonly, accessory functions are provided by infection of the hostcells with an unrelated helper virus. A number of suitable helperviruses are known, including adenoviruses; herpesviruses such as herpessimplex virus types 1 and 2; and vaccinia viruses. Nonviral accessoryfunctions will also find use herein, such as those provided by cellsynchronization using any of various known agents. See, e.g., Buller etal. (1981) J. Virol. 40:241-247; McPherson et al. (1985) Virology147:217-222; Schlehofer et al. (1986) Virology 152:110-117.

Alternatively, accessory functions can be provided using an accessoryfunction vector. Accessory function vectors include nucleotide sequencesthat provide one or more accessory functions. An accessory functionvector is capable of being introduced into a suitable host cell in orderto support efficient AAV virion production in the host cell. Accessoryfunction vectors can be in the form of a plasmid, phage, transposon,cosmid, or another virus. Accessory vectors can also be in the form ofone or more linearized DNA or RNA fragments which, when associated withthe appropriate control elements and enzymes, can be transcribed orexpressed in a host cell to provide accessory functions.

Nucleic acid sequences providing the accessory functions can be obtainedfrom natural sources, such as from the genome of an adenovirus particle,or constructed using recombinant or synthetic methods known in the art.In this regard, adenovirus-derived accessory functions have been widelystudied, and a number of adenovirus genes involved in accessoryfunctions have been identified and partially characterized. See, e.g.,Carter, B. J. (1990) “Adeno-Associated Virus Helper Functions,” in CRCHandbook of Parvoviruses, vol. I (P. Tijssen, ed.), and Muzyczka, N.(1992) Curr. Topics. Microbiol. and Immun 158:97-129. Specifically,early adenoviral gene regions E1a, E2a, E4, VAI RNA and, possibly, E1bare thought to participate in the accessory process. Janik et al. (1981)Proc. Natl. Acad. Sci. USA 78:1925-1929. Herpesvirus-derived accessoryfunctions have been described. See, e.g., Young et al. (1979) Frog. Med.Virol. 25:113. Vaccinia virus-derived accessory functions have also beendescribed. See, e.g., Carter, B. J. (1990), supra., Schlehofer et al.(1986) Virology 152:110-117.

As a consequence of the infection of the host cell with a helper virus,or transfection of the host cell with an accessory function vector,accessory functions are expressed which transactivate the AAV helperconstruct to produce AAV Rep and/or Cap proteins. The Rep expressionproducts excise the recombinant DNA (including the DNA of interest,e.g., the heterologous nucleic acid) from the AAV expression vector. TheRep proteins also serve to duplicate the AAV genome. The expressed Capproteins assemble into capsids, and the recombinant AAV genome ispackaged into the capsids. Thus, productive AAV replication ensues, andthe DNA is packaged into rAAV virions.

Following recombinant AAV replication, rAAV virions can be purified fromthe host cell using a variety of conventional purification methods, suchas CsCl gradients. Further, if infection is employed to express theaccessory functions, residual helper virus can be inactivated, usingknown methods. For example, adenovirus can be inactivated by heating totemperatures of approximately 60° C. for, e.g., 20 minutes or more. Thistreatment effectively inactivates only the helper virus since AAV isextremely heat stable while the helper adenovirus is heat labile.

The resulting rAAV virions are then ready for use for DNA delivery, suchas in gene therapy applications, or for the delivery of a gene productto a mammalian host.

Delivery of a Gene Product

The present invention further provides methods of delivering a geneproduct to an individual in need thereof. The methods generally involveintroducing a subject rAAV virion into an individual.

Generally, rAAV virions are introduced into a cell using either in vivoor in vitro transduction techniques. If transduced in vitro, the desiredrecipient cell will be removed from the subject, transduced with rAAVvirions and reintroduced into the subject. Alternatively, syngeneic orxenogeneic cells can be used where those cells will not generate aninappropriate immune response in the subject.

Suitable methods for the delivery and introduction of transduced cellsinto a subject have been described. For example, cells can be transducedin vitro by combining recombinant AAV virions with cells e.g., inappropriate media, and screening for those cells harboring the DNA ofinterest using conventional techniques such as Southern blots and/orPCR, or by using selectable markers. Transduced cells can then beformulated into pharmaceutical compositions, described more fully below,and the composition introduced into the subject by various techniques,such as by intramuscular, intravenous, subcutaneous and intraperitonealinjection.

For in vivo delivery, the rAAV virions will be formulated intopharmaceutical compositions and will generally be administeredparenterally (e.g., administered via an intramuscular, subcutaneous,intratumoral, transdermal, intrathecal, etc., route of administration.

Pharmaceutical compositions will comprise sufficient genetic material toproduce a therapeutically effective amount of the gene product ofinterest, i.e., an amount sufficient to reduce or ameliorate symptoms ofthe disease state in question or an amount sufficient to confer thedesired benefit. The pharmaceutical compositions will also contain apharmaceutically acceptable excipient. Such excipients include anypharmaceutical agent that does not itself induce the production ofantibodies harmful to the individual receiving the composition, andwhich may be administered without undue toxicity. Pharmaceuticallyacceptable excipients include, but are not limited to, liquids such aswater, saline, glycerol and ethanol. Pharmaceutically acceptable saltscan be included therein, for example, mineral acid salts such ashydrochlorides, hydrobromides, phosphates, sulfates, and the like; andthe salts of organic acids such as acetates, propionates, malonates,benzoates, and the like. Additionally, auxiliary substances, such aswetting or emulsifying agents, pH buffering substances, and the like,may be present in such vehicles. A wide variety of pharmaceuticallyacceptable excipients are known in the art and need not be discussed indetail herein. Pharmaceutically acceptable excipients have been amplydescribed in a variety of publications, including, for example, A.Gennaro (2000) “Remington: The Science and Practice of Pharmacy,” 20thedition, Lippincott, Williams, & Wilkins; Pharmaceutical Dosage Formsand Drug Delivery Systems (1999) H. C. Ansel et al., eds., 7^(th) ed.,Lippincott, Williams, & Wilkins; and Handbook of PharmaceuticalExcipients (2000) A. H. Kibbe et al., eds., 3^(rd) ed. Amer.Pharmaceutical Assoc.

Appropriate doses will depend on the mammal being treated (e.g., humanor nonhuman primate or other mammal), age and general condition of thesubject to be treated, the severity of the condition being treated, theparticular therapeutic protein in question, its mode of administration,among other factors. An appropriate effective amount can be readilydetermined by one of skill in the art.

Thus, a “therapeutically effective amount” will fall in a relativelybroad range that can be determined through clinical trials. For example,for in vivo injection, i.e., injection directly to skeletal or cardiacmuscle, a therapeutically effective dose will be on the order of fromabout 10⁶ to about 10¹⁵ of the rAAV virions, e.g., from about 10⁸ to10¹² rAAV virions. For in vitro transduction, an effective amount ofrAAV virions to be delivered to cells will be on the order of from about10⁸ to about 10¹³ of the rAAV virions. Other effective dosages can bereadily established by one of ordinary skill in the art through routinetrials establishing dose response curves.

Dosage treatment may be a single dose schedule or a multiple doseschedule. Moreover, the subject may be administered as many doses asappropriate. One of skill in the art can readily determine anappropriate number of doses.

In some embodiments, the present invention provides methods ofdelivering a gene product to a stem cell. In these embodiments, asubject rAAV virion is introduced into a stem cell, either in vitro orin vivo. Stem cells of interest include hematopoietic stem cells andprogenitor cells derived therefrom (U.S. Pat. No. 5,061,620); neuralcrest stem cells (see Morrison et al. (1999) Cell 96:737-749); adultneural stem cells or neural progenitor cells; embryonic stem cells;mesenchymal stem cells; mesodermal stem cells; etc. Other hematopoietic“progenitor” cells of interest include cells dedicated to lymphoidlineages, e.g. immature T cell and B cell populations.

Purified populations of stem or progenitor cells may be used. Forexample, human hematopoietic stem cells may be positively selected usingantibodies specific for CD34, thy-1; or negatively selected usinglineage specific markers which may include glycophorin A, CD3, CD24,CD16, CD14, CD38, CD45RA, CD36, CD2, CD19, CD56, CD66a, and CD66b; Tcell specific markers, tumor specific markers, etc. Markers useful forthe separation of mesodermal stem cells include FcγRII, FcγRIII, Thy-1,CD44, VLA-4α, LFA-10, HSA, ICAM-1, CD45, Aa4.1, Sca-1, etc. Neural creststem cells may be positively selected with antibodies specific forlow-affinity nerve growth factor receptor (LNGFR), and negativelyselected for the markers sulfatide, glial fibrillary acidic protein(GFAP), myelin protein P_(o), peripherin and neurofilament. Humanmesenchymal stem cells may be positively separated using the markersSH2, SH3 and SH4.

The cells of interest are typically mammalian, where the term refers toany animal classified as a mammal, including humans, domestic and farmanimals, and zoo, laboratory, sports, or pet animals, such as dogs,horses, cats, cows, mice, rats, rabbits, etc. In some embodiments, thestem cell is a human stem cell.

The cells which are employed may be fresh, frozen, or have been subjectto prior culture. They may be fetal, neonate, adult. Hematopoietic cellsmay be obtained from fetal liver, bone marrow, blood, particularly G-CSFor GM-CSF mobilized peripheral blood, or any other conventional source.The manner in which the stem cells are separated from other cells of thehematopoietic or other lineage is not critical to this invention. Asdescribed above, a substantially homogeneous population of stem orprogenitor cells may be obtained by selective isolation of cells free ofmarkers associated with differentiated cells, while displaying epitopiccharacteristics associated with the stem cells.

Any of a variety of proteins can be delivered to an individual using asubject method. Suitable proteins include, but are not limited to, aninterferon (e.g., IFN-γ, IFN-α, IFN-ω; IFN-τ); an insulin (e.g.,Novolin, Humulin, Humalog, Lantus, Ultralente, etc.); an erythropoietin(“EPO”; e.g., Procrit®, Eprex®, or Epogen® (epoetin-a); Aranesp®(darbepoietin-α); NeoRecormon®, Epogin® (epoetin-β); and the like); anantibody (e.g., a monoclonal antibody) (e.g., Rituxan® (rituximab);Remicade® (infliximab); Herceptin® (trastuzumab); Humira™ (adalimumab);Xolair® (omalizumab); Bexxar® (tositumomab); Raptiva™ (efalizumab);Erbitux™ (cetuximab); and the like), including an antigen-bindingfragment of a monoclonal antibody; a blood factor (e.g., Activase®(alteplase) tissue plasminogen activator; NovoSeven® (recombinant humanfactor VIIa); Factor VIIa; Factor VIII (e.g., Kogenate®); Factor IX;β-globin; hemoglobin; and the like); a colony stimulating factor (e.g.,Neupogen® (filgrastim; G-CSF); Neulasta (pegfilgrastim); granulocytecolony stimulating factor (G-CSF), granulocyte-monocyte colonystimulating factor, macrophage colony stimulating factor, megakaryocytecolony stimulating factor; and the like); a growth hormone (e.g., asomatotropin, e.g., Genotropin®, Nutropin®, Norditropin®, Saizen®,Serostim®, Humatrope®, etc.; a human growth hormone; and the like); aninterleukin (e.g., IL-1; IL-2, including, e.g., Proleukin®; IL-3, IL-4,IL-5, IL-6, IL-7, IL-8, IL-9; etc.); a growth factor (e.g., Regranex®(beclapermin; PDGF); Fiblast® (trafermin; bFGF); Stemgen® (ancestim;stem cell factor); keratinocyte growth factor; an acidic fibroblastgrowth factor, a stem cell factor, a basic fibroblast growth factor, ahepatocyte growth factor; and the like); a soluble receptor (e.g., aTNF-α-binding soluble receptor such as Enbrel® (etanercept); a solubleVEGF receptor; a soluble interleukin receptor; a soluble γ/δ T cellreceptor; and the like); an enzyme (e.g., α-glucosidase; Cerazyme®(imiglucarase; β-glucocerebrosidase, Ceredase® (alglucerase;); an enzymeactivator (e.g., tissue plasminogen activator); a chemokine (e.g.,IP-10; Mig; Groa/IL-8, RANTES; MIP-1α; MIP-1β; MCP-1; PF-4; and thelike); an angiogenic agent (e.g., vascular endothelial growth factor(VEGF) ; an anti-angiogenic agent (e.g., a soluble VEGF receptor); aprotein vaccine; a neuroactive peptide such as bradykinin,cholecystokinin, gastin, secretin, oxytocin, gonadotropin-releasinghormone, beta-endorphin, enkephalin, substance P, somatostatin,prolactin, galanin, growth hormone-releasing hormone, bombesin,dynorphin, neurotensin, motilin, thyrotropin, neuropeptide Y,luteinizing hormone, calcitonin, insulin, glucagon, vasopressin,angiotensin II, thyrotropin-releasing hormone, vasoactive intestinalpeptide, a sleep peptide, etc.; other proteins such as a thrombolyticagent, an atrial natriuretic peptide, bone morphogenic protein,thrombopoietin, relaxin, glial fibrillary acidic protein, folliclestimulating hormone, a human alpha-1 antitrypsin, a leukemia inhibitoryfactor, a transforming growth factor, an insulin-like growth factor, aluteinizing hormone, a macrophage activating factor, tumor necrosisfactor, a neutrophil chemotactic factor, a nerve growth factor a tissueinhibitor of metalloproteinases; a vasoactive intestinal peptide,angiogenin, angiotropin, fibrin; hirudin; a leukemia inhibitory factor;an IL-1 receptor antagonist (e.g., Kineret® (anakinra)); an ion channel,e.g., cystic fibrosis transmembrane conductance regulator (CFTR);dystrophin; utrophin, a tumor suppressor; lysosomal enzyme acida-glucosidase (GAA); and the like. Proteins that can be delivered usinga subject method also include a functional fragment of any of theaforementioned proteins; and functional variants of any of theaforementioned proteins.

In some embodiments, a therapeutically effective amount of a protein isproduced in the mammalian host. Whether a therapeutically effectiveamount of a particular protein is produced in the mammalian host using asubject method is readily determined using assays appropriate to theparticular protein. For example, where the protein is EPO, hematocrit ismeasured.

Where the rAAV encodes an antigenic protein, suitable antigenic proteinsthat can be delivered to an individual using a subject method include,but are not limited to, tumor-associated antigens, autoantigens (“self”antigens), viral antigens, bacterial antigens, protozoal antigens, andallergens; and antigenic fragments thereof. In some embodiments, acytotoxic T lymphocyte (CTL) response to the rAAV-encoded antigenicprotein will be induced in the mammalian host. In other embodiments, ahumoral response to the rAAV-encoded antigenic protein will be inducedin the mammalian host, such that antibodies specific to the antigenicprotein are generated. In many embodiments, a TH1 immune response to therAAV-encoded antigenic protein will be induced in the mammalian host.Whether an immune response to the antigenic protein has been generatedis readily determined using well-established methods. For example, anenzyme-linked immunosorbent assay can be used to determine whetherantibody to an antigenic protein has been generated. Methods ofdetecting antigen-specific CTL are well known in the art. For example, adetectably labeled target cell expressing the antigenic protein on itssurface is used to assay for the presence of antigen-specific CTL in ablood sample.

In some embodiments, e.g., in the context of stem cells, suitableproteins, include, but are not limited to: a growth factor, a morphogen,a cytokine, a receptor, a protein involved in intracellular signaltransduction, a protein that provides a detectable signal, and atranscription factor. For example, in some embodiments, e.g., in thecontext of stem cells, suitable proteins include, but are not limitedto, a growth factor (e.g., epidermal growth factor, fibroblast growthfactor, vascular endothelial growth factor, neurotrophin, a TGFβ familymember, a Delta family member, a Jagged family member); a morphogen(e.g., a Wnt or Hedgehog protein); a cytokine (e.g., an interleukin, atumor necrosis family member, etc.); a receptor (e.g., a growth factorreceptor, a cytokine receptor, a neurotransmitter receptor, aneutrotrophin receptor, an ion channel, a Notch family member, a Patchedfamily member, a TGFβ receptor family member, a steroid hormonereceptor, etc.); a protein involved in intracellular signal transduction(e.g., MAPK, MEKK, PI3-kinase, Akt, PKC, PKA, PKG, a member of the Jakfamily, a member of the Src family, etc.); a transcription factor (e.g.,a member of the GATA, Gli, Sp, Hes, Hey, NF-KB, LIM, Olig, Mash, Math,TCF, Elk, Forkhead, histone acetyltransferase, histonemethyltransferase, or histone deacetylase families); and the like.

In some embodiments, e.g., in the context of lung epithelial cells,suitable proteins include, but are not limited to: a cystic fibrosistransmembrane conductance regulator, an immunogen (e.g., an antigen, asdescribed elsewhere herein), and an antiviral protein. Non-limitingexamples of suitable immunogens include a protein from influenza, aBacillus anthracis polypeptide, a cancer-associated antigen, and otherantigens as described elsewhere herein. In the context of lungepithelial cells, a suitable heterologous nucleic acid comprises anucleotide sequence encoding an antiviral protein (e.g.,interferon-beta, interferon-gamma, interleukin-2, etc.).

Nucleic acids that can be delivered to an individual using a subjectmethod include non-translated RNAs, such as an antisense RNA, aribozyme, an RNAi, an shRNA, and an siRNA. In some embodiments, atherapeutically effective amount of the non-translated RNA is producedin the mammalian host. Whether a therapeutically effective amount of anon-translated RNA has been delivered to a mammalian host using asubject method is readily determined using any appropriate assay. Forexample, where the gene product is an siRNA that inhibits HIV, viralload can be measured.

Methods of Generating Mutant AAV Virions

The present invention provides a method of generating a mutant AAVvirion comprising one or more mutations in one or more of VP1, VP2, andVP3. The method generally involves generating a mutant AAV library; andselecting the library for capsid mutants with altered capsid properties.The present invention further provides mutant AAV libraries, andcompositions comprising the mutant AAV libraries.

In some embodiments, a given selection step is repeated two, three,four, or more times to enrich a subject AAV library for altered capsidproperties. In some embodiments, following selection of an AAV library,individual clones are isolated and sequenced.

Generation of Mutant AAV Library

A mutant AAV library is generated that comprises one or more mutationsin an AAV cap gene. Mutations in the AAV cap gene are generated usingany known method. Suitable methods for mutagenesis of an AAV cap geneinclude, but are not limited to, a polymerase chain reaction (PCR)-basedmethod, oligonucleotide-directed mutagenesis, and the like. Methods forgenerating mutations are well described in the art. See, e.g., Zhao etal. (1998) Nat. Biotechnol. 16:234-235; U.S. Pat. No. 6,579,678; U.S.Pat. No. 6,573,098; and U.S. Pat. No. 6,582,914.

In some embodiments, a mutant AAV library comprising mutations in thecap gene will be generated using a staggered extension process. Thestaggered extension process involves amplification of the cap gene usinga PCR-based method. The template cap gene is primed using specific PCRprimers, followed by repeated cycles of denaturation and very shortannealing/polymerase-catalyzed extension. In each cycle, the growingfragments anneal to different templates based on sequencecomplementarity and extend further. The cycles of denaturation,annealing, and extension are repeated until full-length sequences form.The resulting full-length sequences include at least one mutation in thecap gene compared to a wild-type AAV cap gene.

The PCR products comprising AAV cap sequences that include one or moremutations are inserted into a plasmid containing a wild-type AAV genome.The result is a library of AAV cap mutants. Thus, the present inventionprovides a mutant AAV cap gene library comprising from about 10 to about10¹⁰ members, and comprising mutations in the AAV cap gene. A givenmember of the library has from about one to about 50 mutations in theAAV cap gene. A subject library comprises from 10 to about 10⁹ distinctmembers, each having a different mutation(s) in the AAV cap gene.

Once a cap mutant library is generated, viral particles are producedthat can then be selected on the basis of altered capsid properties.Library plasmid DNA is transfected into a suitable host cell (e.g., 293cells), followed by introduction into the cell of helper virus. Viralparticles produced by the transfected host cells (“AAV libraryparticles) are collected.

Library Selection

Once a library is generated, it is selected for a particular capsidproperty. Viral particles are generated as discussed above, andsubjected to one or more selection steps. Capsid properties that areselected for include, but are not limited to: 1) increased heparinbinding affinity relative to wild-type AAV; 2) decreased heparin bindingaffinity relative to wild-type AAV; 3) increased infectivity of a cellthat is resistant to infection with AAV; 4) increased evasion ofneutralizing antibodies; and 5) increased ability to cross anendothelial cell layer.

1. Selection for Altered Heparin Binding

In some embodiments, a subject library is selected for altered heparinbinding, including increased heparin binding and decreased heparinbinding relative to wild-type AAV virion heparin binding. AAV libraryparticles are contacted with a heparin affinity matrix. For example, AAVlibrary particles are loaded onto a heparin affinity column underconditions that permit binding of the AAV library particles to theheparin. Exemplary conditions include equilibration of the column with0.15 M NaCl and 50 mM Tris at pH 7.5. After allowing the AAV libraryparticle to bind to the heparin affinity matrix, the AAV libraryparticle/heparin affinity matrix complex is washed with volumes ofbuffer containing progressively increasing concentrations of NaCl, andat each NaCl concentration, eluted AAV library particles are collected.For example, after binding the AAV library particle/heparin affinitymatrix complex is washed with a volume of 50 mM Tris buffer, pH 7.5,containing 200 mM NaCl, and eluted AAV library particles are collected.The elution step is repeated with a 50 mM Tris buffer, pH 7.5,containing about 250 mM NaCl, about 300 mM NaCl, about 350 mM, about 400mM NaCl, about 450 mM NaCl, about 500 mM NaCl, about 550 mM NaCl, about600 mM NaCl, about 650 mM NaCl, about 700 mM NaCl, or about 750 mM NaCl.

AAV library particles that elute at NaCl concentrations lower than about450 mM NaCl exhibit decreased heparin binding properties relative towild-type AAV. AAV library particles that elute at NaCl concentrationshigher than about 550 mM NaCl exhibit increased heparin bindingproperties relative to wild-type AAV.

In some embodiments, eluted AAV library particles are amplified byco-infection of permissive cells with a helper virus, and arere-fractionated on heparin affinity matrix. This step can be repeated anumber of times to enrich for AAV library particles with altered heparinbinding properties.

2. Selection for Reduced Binding to Neutralizing Antibodies

In some embodiments, a subject AAV library is selected for reducingbinding to neutralizing antibodies that bind to an neutralize wild-typeAAV virions, compared to the binding of such antibodies to wild-type AAVvirions and neutralization of wild-type AAV virions. AAV libraryparticles are contacted with neutralizing antibodies and the ability ofthe AAV library particles to infect a permissive host cell is tested.Typically, AAV library particles are contacted with variousconcentrations of neutralizing antibodies. The higher the concentrationof neutralizing antibodies that is required to reduce infectivity of theAAV library particles, the more resistant the AAV particles are toneutralization.

3. Selection for Increased Infectivity of Non-Permissive Cells

In some embodiments, a subject AAV library is selected for increasedinfectivity of non-permissive cells. AAV library particles are contactedwith a non-permissive cell (e.g., a population of non-permissive cells).After a suitable amount of time to allow for infection of the cells withAAV library particles, helper virus is added, and AAV library particlesthat successfully infected the non-permissive cell(s) are harvested. Insome embodiments, the cycle of infection, addition of helper virus, andharvesting of AAV particles is repeated one, two, three, or more times.

In the present methods, one or more selection steps may followgeneration of AAV library particles. For example, in some embodiments,the method comprises selecting for increased heparin binding, followedby selecting for decreased binding to neutralizing antibodies. In otherembodiments, the method comprises selecting for decreased binding toneutralizing antibodies, followed by selecting for increased heparinbinding. In other embodiments, the method comprises selecting fordecreased heparin binding, followed by selecting for decreased bindingto neutralizing antibodies. In other embodiments, the method comprisesselecting for decreased binding to neutralizing antibodies, followed byselecting for decreased heparin binding. In other embodiments, themethod comprises selecting for decreased binding to neutralizingantibodies, followed by selecting for increased infectivity of a stemcell.

Thus, the present invention provides an adeno-associated virus (AAV)library, that includes a plurality of nucleic acids, each of whichnucleic acids include a nucleotide sequence that encodes a mutant AAVcapsid protein. The encoded mutant AAV capsid protein includes at leastone amino acid substitution relative to a wild-type AAV capsid protein.The present invention provides a library of mutant adeno-associatedvirus (AAV) particles, including a plurality of AAV particles each ofwhich includes an AAV capsid protein that includes at least one aminoacid substitution relative to a wild-type AAV capsid protein. Nucleicacids encoding mutant AAV capsid proteins are described above, as arethe properties of the encoded mutant AAV capsid proteins.

EXAMPLES

The following examples are put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how tomake and use the present invention, and are not intended to limit thescope of what the inventors regard as their invention nor are theyintended to represent that the experiments below are all or the onlyexperiments performed. Efforts have been made to ensure accuracy withrespect to numbers used (e.g. amounts, temperature, etc.) but someexperimental errors and deviations should be accounted for. Unlessindicated otherwise, parts are parts by weight, molecular weight isweight average molecular weight, temperature is in degrees Celsius, andpressure is at or near atmospheric. Standard abbreviations may be used,e.g., bp, base pair(s); kb, kilobase(s); pl, picoliter(s); s, second(s);min, minute(s); hr, hour(s); and the like.

Example 1 Generation and Characterization of AAV Capsid Variants MethodsLibrary Generation and Vector Packaging

An AAV2 cap ORF genetic library was generated using the staggeredextension process described by Zhao at al. ((1998) Nat. Biotechnol.16:258-261), and the resulting cap product was inserted into a plasmidcontaining the wild type AAV2 genome. The result was transformed into E.coli for large scale plasmid production and purification. AAV was thenproduced and purified by CsCl centrifugation essentially as previouslydescribed (Kaspar et al. (2002) Proc. Natl. Acad. Sci. USA 99:2320-2325;and Lai et al. (2003) Nat. Neurosci. 6:21-27). Briefly, the libraryplasmid DNA was transfected into 293 human embryonic kidney cells (ATCC)using the calcium phosphate method, followed by addition of serotype 5adenovirus (Ad5) at a multiplicity of infection (MOI) of 3. Virus waspurified using CsCl density centrifugation. For all experiments, the AAVgenomic titer was determined by extracting vector DNA as previouslydescribed (Kaspar et al. supra; and Lai et al., supra) followed byquantification using real time PCR with SYBR-Green dye (MolecularProbes) and a Biorad iCycler.

Heparin Column Chromatography

Approximately 10¹² AAV library particles were loaded onto a 1 mL HiTrapheparin column (Amersham) previously equilibrated with 0.15 M NaCl and50 mM Tris at pH 7.5. Washes were performed using 0.75 ml volumes of thesame buffer with increasing increments of 50 mM NaCl up to 750 mM,followed by a 1 M wash. As a control, rAAV-GFP was also subjected toheparin affinity chromatography. To isolate individual viral clones fromlibrary fractions that eluted at different salt concentrations, viralDNA was extracted from the fractions, amplified by PCR, and insertedinto a rAAV packaging plasmid based on pAd8. rAAV-GFP was then packagedin order to analyze the ability of these mutant capsids to package rAAVvector. Capsid variants were sequenced at the U.C. Berkeley DNAsequencing facility. Finally, the affinity of the variant capsids forheparin was quantified by using the method of Qiu et al. ((2000) Virol.269:137-147), except that virus bound to the immobilized heparin wasquantified by real time PCR.

Antisera Generation and Antibody Neutralization Screen

Polyclonal sera containing neutralizing antibodies (NABs) against AAV2were generated in two New Zealand White rabbits in accordance with theU.C. Berkeley Animal Care and Use Committee and NIH standards forlaboratory animal care. Briefly, 5×10¹⁰ CsCl-purified rAAV2 particleswere mixed with 0.5 mL TitreMax adjuvant (CytRx) and injected into theanterior hindlimb muscle. Two boosts were performed at 3-week intervalsusing the same AAV dosage, followed by antiserum collection.

Both wild type and a mutant AAV library were incubated with varyingamounts of serum (0-6.25 μl) in 75 μl of phosphate-buffered saline (PBS)(pH 7.4) for 30 minutes at room temperature, followed by addition to2.5×10⁵ 293 cells in a 6-well format. After 48 hours, AAV was rescuedfrom infected cells by addition of Ad5, and cells were harvested 24hours later.

Individual viral clones from the library fraction that successfullyinfected cells even in the presence of NAB were inserted into the rAAVpackaging plasmid, and rAAV-GFP was produced as above. rAAV with mutantcapsids were then incubated in 5 μl polyclonal sera as above, followedby addition to 1×10⁵ 293 cells. At 72 hours post-infection, the fractionof green cells was quantified by flow cytometry at the U.C. BerkeleyCancer Center (Beckman-Coulter EPICS).

Results Library Generation

The staggered extension process (Zhao et al., supra), a polymerase chainreaction (PCR)-based method that generates diverse genetic libraries ina manner similar to that of DNA shuffling, was used to generate alibrary of cap mutants with point mutations randomly distributedthroughout VP1-3. This product was inserted into a plasmid containingthe complete wild type AAV genome to yield a viral library withapproximately 10⁶ independent clones, as determined by quantifying thenumber of colonies following bacterial transformation. To assess itsdegree of sequence diversity, the plasmid library was sequenced. Theplasmid was then packaged into virus by transient transfection into 293cells followed by Ad5 infection to yield a viral particle library. Thislarge AAV library is selected for viral variants with any variety of newproperties or functions, and the capsid structure conferring these newfunctions are readily recovered by DNA sequence analysis of the AAVgenome encapsidated in the particles.

Heparin Binding Mutants

As an initial gauge of how the library's sequence diversity translatedinto capsid functional diversity, CsCl-purified library particles weresubjected to heparin affinity chromatography with steps of increasingNaCl concentration. As previously reported (Zolotukhin et al. (1999)Gene Ther. 6:973-985) wild type AAV elutes from heparin between 450 and550 mM NaCl (FIG. 1). In stark contrast, the AAV mutant library elutesat a wide range of salt concentrations, from the 150 mM load to thefinal 750 mM fraction. This result demonstrates that the libraryencompasses significant sequence and functional diversity. Since itsaffinity for heparan sulfate limits the spread of rAAV2 vectors uponinjection in vivo, lower affinity mutants may be desirable as genedelivery vectors when wide dissemination through a large tissue orregion is needed. In contrast, a higher affinity mutant may beadvantageous for regionally pinpointed, high level gene expression.

FIGS. 1a and 1b . Heparin binding characteristics of wild type AAV vs.the viral library. a) The heparin affinity column chromatogram ofelution of wild type AAV (hatched bars) and the mutant library (openbars) is shown. Virus gradually elutes from the column as the NaClconcentration is increased. b) Chromatograms of pools from the mutantlibrary selected for lower (hatched bars) and higher (open bars) heparinaffinities.

To isolate mutants with both low and high heparin affinity, the 150 mMand 700 mM NaCl fractions from the initial library were separatelyamplified by co-infection of 293 cells with Ad5, and refractionated onthe heparin column. After three rounds of enrichment, the majority ofthe two resulting viral pools eluted from the column at 150 mM and 750mM (FIG. 1b ). Importantly, since each round of enrichment involved 293cell infection, these pools are still composed of highly infectiousvirus. Next, individual capsid clones were isolated from each of thesesalt fractions and sequenced.

The fact that these mutants eluted from the heparin column at differentsalt concentrations indicates that they had different affinities forheparin. To accurately measure these affinities, however, the method ofQiu et al supra can be used. By performing Scatchard binding analysis ofvirus to heparin immobilized to microtiter plates, the difference in theaffinity of the mutants for heparin, compared to the affinity ofwild-type AAV for heparin, is determined.

Neutralizing Antiserum Escape Mutants

The isolation of functionally diverse heparin binding mutants from ourAAV mutant library demonstrates the utility of this approach forcreating vectors with novel properties. This approach was applied to amuch more significant problem: vector elimination by neutralizingantibodies. Rabbit anti-AAV2 neutralizing antiserum was generated, and a1:1500 serum dilution was sufficient to inhibit rAAV-GFP gene deliveryto 293 cells by 50%, comparable to NAB titers in human serum.

FIGS. 2a and 2b . Generation of antibody neutralization escape mutants.a) Virus was incubated with antiserum before addition to 293 cells. Thelevel of virus that successfully evaded antibody neutralization ascompared to control infection in the absence of antiserum was measuredby addition of Ad5 and titering of rescued AAV by quantitative real timePCR on AAV genomes. b) The data on individual variants is presented.

Antibody escape mutants were isolated from the viral library. Duringsuccessive rounds of selection, the library was subjected to increasingstringencies, where stringency is defined as the ratio of the dilutionnecessary to reduce rAAV2 with wild type capsid by 50% (a 1500-fold forthe rabbit antiserum) to the dilution actually used. Therefore, the moreantiserum added to the virus, the higher the stringency. After threerounds of selection, followed by co-infection of 293 cells with Ad5 foramplification, the resulting viral pool was less susceptible to antibodyneutralization as compared to wild type virus. Next, five individualcapsid clones from this pool were analyzed by using them to packagerAAV-GFP. The results demonstrated that the resulting recombinant vectoris between 100 and 1000-fold more resistant to neutralizing antibodies.This is a therapeutically relevant result.

Neutralizing antibody inhibition of rAAV gene delivery can be observedas follows. rAAV2 with wild type capsid is added to 293 cells andvisualized after 24 hours. Virus is visualized in green (Alexa 488),microtubules in red (Alexa 546), and the nucleus in blue (TO-PRO-3).After preincubation in neutralizing antiserum, the intracellulartransport of rAAV2 with wild type capsid is expected to be significantlyinhibited.

In FIGS. 3A-C, the cap nucleotide sequences of two neutralizing antibodyescape mutants is shown, compared to wild-type cap sequence.

Example 2 Characterization of Further AAV Capsid Variants

AAV mutants—including antibody evaders, mutants with reduced heparinbinding, and mutants with increased heparin binding—were generated asfollows.

Library Generation:

A combination of error prone PCR and the staggered extension process(StEP) was used to generate a library of mutant DNA fragments encodingthe capsid protein. This library was then inserted into a plasmid inorder to package it into virus, by transfection of these plasmids plusan adenoviral helper plasmid into 293 cells. The library was thenpurified by cesium chloride density centrifugation.

Selection:

The library was then passed through screening steps. For example, forthe antibody evasion mutant, a sample of the library was mixed togetherwith polyclonal anti-AAV antiserum, then added to 293 cells. Addition ofadenovirus amplifies the variants that are able to successfully evadethe antiserum.

After amplification of the ‘successful’ variants (e.g., variants thatinfected the 293 cells in the presence of anti-AAV antibody) in 293cells, the variants were purified, mixed again with antiserum, andallowed to infect 293 cells, and finally reamplified with adenovirus tofurther enrich for variants. After several rounds of enrichment, theviral DNA was purified, cloned, and sequenced to determine whichmutations resulted in successful variants.

Generating Vector:

At this point, only the viral capsid DNA had been used to packagereplication competent virus, i.e. viruses with a genome that containedrep and the mutant cap genes and were therefore capable of replicatingin the presence of adenovirus. However, to use the variants in a genetherapy setting, it was important to use the mutant cap gene to packagerecombinant particles containing a therapeutic gene. To demonstrate thatthe mutant cap genes described herein are capable of packagingrecombinant virus, the cap gene was moved into a packaging/helperplasmid. Addition of this helper along with an adenoviral helper plasmidand a vector plasmid containing a promoter driving the expression of areporter gene (GFP) to cells results in the generation of recombinantparticles.

Antibody Evasion Mutants

Two sources of antiserum were used to generate antibody evasion mutants.One is rabbit serum that was generated by injecting rabbits with wildtype AAV particles (Anti-AAV Rabbit Serum produced from New ZealandWhite rabbits). The second is human serum pooled from a number ofindividuals (Sigma Product #H-1388; Lot #122K0424; Origin: 40 NorthAmerican donors).

Heparin Binding Mutants

To generate heparin binding mutants, CsCl-purified library viralparticles were subjected to heparin affinity chromatography with stepsof increasing NaCl concentration, as described in Example 1. Mutantswith reduced heparin binding were eluted with the same NaClconcentration used to load the viral particles (e.g., 0.15 M NaCl).Mutants with reduced heparin binding were eluted at between 650-700 mMNaCl.

Characterization

Tables 1-4, below, provide the location of the amino acid differences inVP1 from wild-type AAV VP1 for the various mutants. All amino aciddifferences are relative to the VP1 amino acid sequence shown in FIGS.5A-C (SEQ ID NO:5). VP1-encoding nucleotide sequence of the variousmutants are provided in FIGS. 4A-G, FIGS. 6A-J, and FIGS. 8A-G. In FIGS.4A-G, FIGS. 6A-J, and FIGS. 8A-G, nucleotide differences from wild-typeAAV-2 VP1 are shown in bold. VP1 amino acid sequences of the variousmutants are provided in FIGS. 5A-C, FIGS. 7A-D, and FIGS. 9A-C. In FIGS.5A-C, FIGS. 7A-D, and FIGS. 9A-C, conservative amino acid changes areindicated with a box; and non-conservative amino acid changes areindicated in bold.

Table 1 provides the clone number and amino acid changes for various AAVneutralizing antibody escape mutants.

TABLE 1 Mutations in 5 rabbit antisera evader clones Clone MutationRegion rAbE1 T713R 2-fold dimple* T716A 2-fold dimple* rAbE2 V418LAntigenic peptide 53* T713R 2-fold dimple* T716A 2-fold dimple* rAbE3T716A 2-fold dimple* rAbE4 D180N A69 linear epitope^(†) T716A 2-folddimple* rAbE5 A493G C37-B conformational epitope^(†) T716A 2-folddimple* Moskalenko et al. (2000) J. Virol. 74: 1761-1766 ^(†)Wobus etal. (2000) J. Virol. 74: 9281-9293 ^(‡)Xie et al. (2002) Proc. Natl.Acad. Sci. USA 99: 10405-10410

Table 2 provides the clone number and amino acid changes for various AAVneutralizing antibody escape mutants.

TABLE 2 Mutations in 9 human antisera evader clones Clone MutationRegion S1CM5 K169R Peptide 22-23* & Bordering A69 linear epitope^(†)S181P A69 linear epitope^(†) A333V 5-fold cylinder* P363T Between 5-foldcylinder and A20 conformational epitope A493E C37-B conformationalepitope^(†) S2CM5 I19V ″Lip″ insertion (Peptide 4-5)* V369A A20conformational epitope^(†) A593E Accessible surface region in Loop 4 onβ-GH13^(‡) S2CM2 G189E 6 aa proximity to A69 linear epitope^(†) N215INear N-terminus VP3 A367E Between 5-fold cylinder and A20 conformationalepitope, 2 aa proximity to A20 S429G α-GH1^(‡) A493E C37-Bconformational epitope^(†) S580P End of b-GH12, 1 aa proximity toaccessible surface region loop 4, β-GH13^(‡) P643L Proline residuebetween β-GH16 and β-H in β barrel core^(‡) E685V S3CM2 R20S ″Lip″insertion (Peptide 4-5)* N57S E347V 5-fold cylinder* A493E C37-Bconformational epitope^(†) N551D D594E Accessible surface region in Loop4 on β-GH13^(‡) A1CM5 K26R ″Lip″ insertion (Peptide 4-5)* N215D G35555-fold cylinder* A593E Accessible surface region in Loop 4 on β-GH13^(‡)A3CM5 G49E S196P T337P 5-fold cylinder* A1CM2 K24E ″Lip″ insertion(Peptide 4-5)* L91P K137T Q186L 3 aa proximity A69 linear epitope* T251AMinor Peptide 33, Canyon Epitope* H290L F306L End of 5-fold cylinder*S390T A493E C37-B conformational epitope^(†) A505V 1 aa proximity toC37-B conformational epitope^(†) V557I T651A A2CM2 V46A S196P BetweenA20 and C37 minor Conformational Epitope^(†) A593E Accessible surfaceregion in Loop 4 on β-GH13^(‡) A3CM2 P31L F100S I260T Minor Peptide 33,Canyon Epitope* R459G 3-fold spike in Loop 3 (peptide 58)* A522T A663VE681G W694R

Table 3 provides the clone number and amino acid changes for various AAVmutants with increased heparin binding.

TABLE 3 Mutations of Clones with Increased Heparin Affinity CloneMutation D14H1 W23L D231G S261F V323F Q349P G406E N408D D14L3 W23L S196TD231G S261F Q349P G406E N408D N569D N596D

Table 4 provides the clone number and amino acid changes for various AAVmutants with reduced heparin binding.

TABLE 4 Mutations of Clones with Reduced Heparin Affinity Clone MutationP1BH1 M235V Q401R L437H N582D T660A P1BH2 G111R K258E L315P E322G N551DV605I

Example 3 Infectious Virus Exhibiting Infectivity of Lung EpithelialCells

To develop better treatments for cystic fibrosis (CF) and other airwaydisorders, AAV was evolved to be more infectious for human lung tissue,e.g., for lung epithelial cells.

Methods Selection of AVV Library Using Well-Differentiated Human AirwayEpithelia

Two divergent serotypes that utilize distinct receptors, AAV2 and AAV5were combined, by subjecting the cap genes encoding the viral capsomeresto DNA shuffling and error-prone PCR, to yield a highly diverse libraryof ˜10⁶ independent chimeric viral sequences. Species-specificdifferences in various cell types, particularly airway epithelia, havebeen shown to impact viral infection; therefore, to evolve the virustowards clinical gene therapy, a well-differentiated organotypic humanairway model was employed. Specifically, extensive selections wereperformed on human airway epithelial cultures (15 donors) by apicalapplication of the AAV viral library, followed by amplification viabasolateral application of helper wild-type adenovirus. AAV library wasapplied apically for decreasing times and multiplicity of infection(MOI) over 5 rounds (FIG. 11a ). Additional diversification (A) wasperformed after round 3.

Apical transduction by the novel AAV chimera, several correspondingvariants, and serotypes 2, 5, and 9, harboring luciferase was monitoredover a twenty-eight day time course. Quantitative expression wasdetermined after d-luciferin addition to each culture by IVIS imaging(Xenogen).

Characterization of Cystic Fibrosis Transmembrane Conductance Regulator(CFTR) Expressed by AAV Variants.

To investigate whether AAV2.5T could efficiently express CFTR andcorrect the chloride transport defect, cystic fibrosis airway epitheliawere transduced with AAV2.5T encoding CFTR at a MOI of 50,000. Thirtydays post-transduction, epithelia were analyzed in Ussing chambers aspreviously described (Ostedgaard, L.Sl. et al. (2005) Proc Natl Acad SciUSA 102:2952-7).

Results Efficient Variants for Pulmonary Gene Delivery

With every round of selection, increasing amounts of viral progeny wererecovered relative to wild-type AAV2. AAV-luciferase expression peakedat day 21 post-transduction. By round 5, recovery of the evolved progenywas ˜550-fold higher than AAV2 (FIG. 11a ). Sequencing of eight randomclones from the progeny revealed a single AAV variant, AAV2.5T (SEQ IDNO:42), which includes several amino acid differences from AAV2,including a point mutation at amino acid 581 (A581T). Alignment of thisvariant with wild-type AAV2 capsid is shown in FIG. 10A-B.

Strikingly, twenty-one days post-transduction, AAV2.5T-Luc outperformedAAV2-Luc (100-fold), AAVS-Luc (10-fold), and AAV9-Luc (20-fold) (FIG.11b ). No difference in basolateral transduction was observed (FIG. 11e), indicating that the advantage of AAV2.5T was specific for the apicalsurface.

FIGS. 11A-E depict the infectivity and the transduction of AVV vectorsfor pulmonary gene delivery in well-differentiated human airwayepithelia. A) AAV library was applied apically for decreasing times andmultiplicity of infection (MOI) over 5 rounds. AAV-luciferase expressionafter apical B) or basolateral C) transduction was monitored over 28days and peaked at day 21 post-transduction. E) Cystic fibrosis (CF)epithelia lack cAMP-regulated chloride transport. F) AAV2.5T-CFTR (MOI50,000) corrected cAMP-regulated chloride current.

Binding and Transduction of Variant AAV

Recombinant AAV2.5T bound to the apical surface significantly betterthan AAV5 (100-fold, FIG. 12a ), and in contrast to AAV5, binding ofAAV2.5T did not saturate at doses ranging from 10 to 1000 genomecopies/cell (vg/cell). This suggested that the number of viral receptorsand possibly binding affinity were increased. AAV2.5T mirrors parentalAAV5 sensitivity for sialic acid and does not efficiently transducesialic acid deficient Lec2 cells whereas transduction is not altered onheparan sulfate mutants pgsA or pgsD.

Furthermore, AAV2.5T binding to the apical surface of human airwayepithelia is significantly reduced by pretreatment with neuraminidase(p<0.001) (FIG. 12c ). In contrast to airway epithelia, similartransduction was observed between AAV2.5T and AAV5 in several cell linesand primary human astrocytes (FIG. 12d ). Hence, transduction studies onother cell types indicate the advantage of AAV2.5T is cell-type specificand that AAV2.5T requires sialic acid for efficient transduction.

Interestingly, when the mutation (AAV5-A581T) and chimera (AAV2.5) werestudied independently, neither was better than AAV5 at binding ortransduction (FIG. 12). The A581T mutation occurs in a region criticalto AAV5 sialic acid binding. Therefore, a mutation in this region maypotentially influence the binding affinity for sialic acid, the type oflinkages recognized, and/or species specificity. Similar effects havebeen observed for single mutations in other parvoviruses and influenza.However, the AAV5-A581T virus produced low genomic titers and failed tobind or transduce airway epithelia (FIG. 11b ). Likewise, AAV2.5 offeredno advantage over AAV5 in airway epithelia, even though aa1-128 fromAAV2 may potentially alter intracellular trafficking. In total, our datasuggest that the recombination event rescues a structurally deleteriousyet functionally advantageous mutation (A581T).

FIGS. 12 A-D depict binding and cell specificity of an exemplary mutantinfectious for lung tissue. A) AAV2.5T binding does not saturate atdoses between 300 and 1,000 viral genomes per cell (vg/cell). AAV2.5Tmirrors parental AAV5 sensitivity for sialic acid and does notefficiently transduce sialic acid deficient Lec2 cells whereas B)transduction is not altered on heparan sulfate mutants pgsA or pgsD. C)AAV2.5T binding to the apical surface of human airway epithelia issignificantly reduced by pretreatment with neuraminidase (p<0.001). D)In contrast to airway epithelia, similar transduction was observedbetween AAV2.5T and AAV5 in several cell lines and primary humanastrocytes.

Characterization of CFTR Expressed by AAV Variants

CF epithelia did not transport chloride via CFTR, as shown by the lackof change in current after treatment with IBMX/Forskolin (ΔIsc_(cAMP)0±0 μA.cm⁻²) or with the CFTR blocker GlyH-101 (ΔIsc_(GlyH) 0±0 μA.cm⁻²)(FIG. 11d ). CFTR was undetectable by immunocytochemistry. In starkcontrast, AAV2.5T-CFTR restored CFTR chloride current (ΔIsc_(cAMP) 12±4μA.cm⁻², ΔIsc_(GlyH) 18±9 μA.cm⁻²) (FIG. 11e ), and readily-detectableCFTR protein was properly localized at the apical membrane of theepithelial cells. In addition, immunostaining of normal airway epitheliafailed to detect CFTR, suggesting that even lower MOIs may be sufficientfor chloride transport correction.

Example 4 AAV Capsid Variants Conferring Increased Infectivity of HumanEmbryonic Stem Cells

AAV virions with variant capsid proteins were generated as describedabove. Variants that exhibit increased infectivity of human ES cells,compared to the infectivity of wild-type AAV for human ES cells, wereselected. The viral library was added to human embryonic stem cell linehSF6, followed by the addition of wild type adenovirus type 5 to inducereplication of AAV variants that successfully infected the cells. Thecells were then lysed to harvest the virus, and the AAV sequences wererecovered by PCR and reinserted into an AAV genome plasmid. The selectedAAV library was then repackaged. After three such selection steps, i.e.infection and amplification, the viral capsid encoding sequence wassubjected to additional genetic diversification by error prone PCR.

Amino acid sequences of capsid proteins of variants produced after afirst and a second round of evolution were determined, where each roundof evolution was composed of genetic diversification and three selectionsteps. Infectivity of human ES (hES) cells of the first-round variantsis shown in FIG. 13 Amino acid sequences of capsid proteins offirst-round variants are shown in FIGS. 14A-D. Infectivity (expressed as% GFP positive cells) of hES cells of the second-round variants is shownin FIG. 15 Amino acid sequences of capsid proteins of first-roundvariants are shown in FIGS. 16A-C.

As shown in FIG. 13, a number of AAV variants exhibited infectivity ofhES cells that is greater than the infectivity of wild-type AAV (e.g.,AAV2, AAV4, AAV6) for these cells. In addition, as shown in FIG. 15, thevariants designated hEr2.4 and hEr2.29 exhibited greater infectivity ofhES cells (expressed as % GFP positive cells), compared to AAV2. It isnoted that the variant designated hEr2.29 and the variant designatedhEr1.23 have the same capsid amino acid sequence.

FIGS. 17A and 17B provide an amino acid sequence alignment of the aminoacid sequences of hEr2.4, hEr1.23, and hEr3.1 capsids, compared withthat of AAV2 capsid. FIG. 18 shows infectivity of hES cells (representedas % GFP-positive) for hEr1.23, hEr2.4, and hEr3.1, compared with thatof wild-type AAV1, AAV2, AAV4, AAVS, AAV8, and AAV9. The data aresummarized in Table 5, below.

TABLE 5 Avg Std 1 2 3 AAV1 4.48333 0.37072 4.84 4.1 4.51 AAV2 9.5766670.697878 10.08 8.78 9.87 AAV4 0.64 0.105357 0.53 0.74 0.62 AAV5 0.3533330.160728 0.42 0.47 0.17 AAV5 0/683333 0.155349 0.51 0.81 0.73 AAV90.763333 0.126623 0.74 0.9 0.65 hEr1.23 25.29667 1.815746 23.99 27.3724.53 hEr2.4 15.20333 3.082764 16.68 11.66 17.27 hEr3.1 32.86 3.7499230.71 30.68 37.19

While the present invention has been described with reference to thespecific embodiments thereof, it should be understood by those skilledin the art that various changes may be made and equivalents may besubstituted without departing from the true spirit and scope of theinvention. In addition, many modifications may be made to adapt aparticular situation, material, composition of matter, process, processstep or steps, to the objective, spirit and scope of the presentinvention. All such modifications are intended to be within the scope ofthe claims appended hereto.

What is claimed is:
 1. A recombinant adeno-associated virus (rAAV)virion comprising: a) a variant AAV capsid protein, wherein the variantAAV capsid protein comprises at least one amino acid substitutionrelative to a corresponding parental AAV capsid protein, and wherein thevariant capsid protein confers increased infectivity of a stem cellcompared to the infectivity of the stem cell by an AAV virion comprisingthe corresponding parental AAV capsid protein; and b) a heterologousnucleic acid comprising a nucleotide sequence encoding a gene product.2. The rAAV virion of claim 1, wherein the rAAV virion exhibits at least5-fold increased infectivity of a stem cell compared to the infectivityof the stem cell by an AAV virion comprising the corresponding parentalAAV capsid protein.
 3. The rAAV virion of claim 1, wherein the rAAVvirion exhibits at least 10-fold increased infectivity of a stem cellcompared to the infectivity of the stem cell by an AAV virion comprisingthe corresponding parental AAV capsid protein.
 4. The rAAV virion ofclaim 1, wherein the rAAV virion exhibits at least 25-fold increasedinfectivity of a stem cell compared to the infectivity of the stem cellby an AAV virion comprising the corresponding parental AAV capsidprotein.
 5. The rAAV virion of claim 1, wherein the rAAV virion exhibitsat least 50-fold increased infectivity of a stem cell compared to theinfectivity of the stem cell by an AAV virion comprising thecorresponding parental AAV capsid protein.
 6. The rAAV virion of claim1, wherein the variant capsid protein comprises from one to 15 aminoacid sequence substitutions compared to the amino acid sequence of anAAV2 capsid protein set forth in SEQ ID NO:2.
 7. The rAAV virion ofclaim 6, wherein the variant capsid protein comprises an amino acidsequence having at least about 85% amino acid sequence identity to theamino acid sequence set forth in SEQ ID NO:53 (hEr2.29 or hEr1.23). 8.The rAAV virion of claim 6, wherein the variant capsid protein comprisesan amino acid sequence having at least about 85% amino acid sequenceidentity to the amino acid sequence set forth in SEQ ID NO:54 (hEr2.4).9. The rAAV virion of claim 6, wherein the variant capsid proteincomprises an amino acid sequence having at least about 85% amino acidsequence identity to the amino acid sequence set forth in SEQ ID NO:59(hEr3.1).
 10. The rAAV virion of claim 1, wherein gene product is aninterfering RNA.
 11. The rAAV virion of claim 1, wherein the geneproduct is a polypeptide.
 12. The rAAV virion of claim 1, wherein theparental AAV capsid protein is wild-type AAV2 capsid protein.
 13. TherAAV virion of claim 1, wherein the parental AAV capsid protein iswild-type AAVS capsid protein.
 14. A method of delivering a gene productto a stem cell in an individual, the method comprising administering tothe individual a recombinant adeno-associated virus (rAAV) virionaccording to claim
 1. 15. The method of claim 14, wherein the geneproduct is a protein.
 16. The method of claim 14, wherein the geneproduct is a short interfering RNA.
 17. The method of claim 15, whereinthe protein is a growth factor, a morphogen, a cytokine, a receptor, aprotein involved in intracellular signal transduction, or atranscription factor.
 18. A recombinant adeno-associated virus (rAAV)virion comprising: a) a variant AAV capsid protein, wherein the variantAAV capsid protein comprises at least one amino acid substitutionrelative to a corresponding parental AAV capsid protein, and wherein thevariant capsid protein confers increased infectivity of a lungepithelial cell compared to the infectivity of the lung epithelial cellby an AAV virion comprising the corresponding parental AAV capsidprotein; and b) a heterologous nucleic acid comprising a nucleotidesequence encoding a gene product.
 19. The rAAV virion of claim 18,wherein the rAAV virion exhibits at least 5-fold increased infectivityof a lung epithelial cell compared to the infectivity of the lungepithelial cell by an AAV virion comprising the corresponding parentalAAV capsid protein.
 20. The rAAV virion of claim 18, wherein the rAAVvirion exhibits at least 10-fold increased infectivity of a lungepithelial cell compared to the infectivity of the lung epithelial cellby an AAV virion comprising the corresponding parental AAV capsidprotein.
 21. The rAAV virion of claim 18, wherein the rAAV virionexhibits at least 25-fold increased infectivity of a lung epithelialcell compared to the infectivity of the lung epithelial cell by an AAVvirion comprising the corresponding parental AAV capsid protein.
 22. TherAAV virion of claim 18, wherein the rAAV virion exhibits at least50-fold increased infectivity of a lung epithelial cell compared to theinfectivity of the lung epithelial cell by an AAV virion comprising thecorresponding parental AAV capsid protein.
 23. The rAAV virion of claim18, wherein the variant capsid protein comprises from one to 45 aminoacid sequence substitutions compared to the amino acid sequence of anAAVS capsid protein set forth in SEQ ID NO:43.
 24. The rAAV virion ofclaim 18, wherein the variant capsid protein comprises from an A581Tsubstitution compared to the amino acid sequence of an AAVS capsidprotein set forth in SEQ ID NO:43.
 25. A method of delivering a geneproduct to a lung epithelial cell in an individual, the methodcomprising administering to the individual a recombinantadeno-associated virus (rAAV) virion according to claim
 17. 26. Themethod of claim 25, wherein the gene product is a protein.
 27. Themethod of claim 25, wherein the gene product is a short interfering RNA.28. The method of claim 26, wherein the protein is a cystic fibrosistransmembrane conductance regulator, an immunogen, or an antiviralprotein.